Distribution and Function of Carbonic Anhydrase Related Proteins (Carps) VIII, X and XI

ASHOK ASPATWAR

Distribution and Function of Carbonic Anhydrase Related Proteins (CARPs) VIII, X and XI

ACADEMIC DISSERTATION To be presented, with the permission of the Board of the BioMediTech of the University of Tampere, for public discussion in the Main Auditorium of Building B, School of Medicine of the University of Tampere, Medisiinarinkatu 3, Tampere, on October 31st, 2014, at 12 o’clock.

UNIVERSITY OF TAMPERE ASHOK ASPATWAR

Distribution and Function of Carbonic Anhydrase Related Proteins (CARPs) VIII, X and XI

Acta Universitatis Tamperensis 1981 Tampere University Press Tampere 2014 ACADEMIC DISSERTATION University of Tampere, BioMediTech Finland

Supervised by Reviewed by Professor Mauno Vihinen Professor Matti Airaksinen University of Lund University of Helsinki Sweden Finland Professor Seppo Parkkila Professor Todd Alexander University of Tampere University of Alberta Finland Canada

The originality of this thesis has been checked using the Turnitin OriginalityCheck service in accordance with the quality management system of the University of Tampere.

Cover design by Mikko Reinikka

Distributor: [email protected] http://granum.uta.fi

Acta Universitatis Tamperensis 1981 Acta Electronica Universitatis Tamperensis 1468 ISBN 978-951-44-9594-6 (print) ISBN 978-951-44-9595-3 (pdf) ISSN-L 1455-1616 ISSN 1456-954X ISSN 1455-1616 http://tampub.uta.fi

Suomen Yliopistopaino Oy – Juvenes Print

Tampere 2014 441 729 Painotuote Contents

Contents ...... 3

List of original communications ...... 6

Abbreviations ...... 7

Abstract ...... 9

1 Introduction ...... 11

2 Review of the literature ...... 12

2.1 Carbonic anhydrases ...... 12 2.1.1 Historical aspects of carbonic anhydrase isozymes...... 12

2.2 Carbonic anhydrase related proteins ...... 12

2.3 General properties of CARP VIII ...... 15 2.3.1 Expression of CARP VIII in animal models ...... 15 2.3.2 Expression of CARP VIII in humans and human cell line ...... 16 2.3.3 Crystal structure of CARP VIII and its interaction with ITPR1 ...... 17 2.3.4 Role of Car8 gene in gait disorder in mouse ...... 19 2.3.5 Association of CA8 gene with ataxia in humans ...... 19 2.3.6 Role of CARP VIII in cancer and other diseases ...... 21 2.3.7 Catalytic activity and inhibition of mutant CARP VIII ...... 23

2.4 General properties of CARP X ...... 24 2.4.1 Expression of CARP X in human and mouse tissues ...... 24 2.4.2 Expression of CARP X in pathological conditions ...... 25 2.4.3 Catalytic activity and inhibition of mutant CARP X ...... 26

2.5 General properties of CARP XI ...... 26 2.5.1 Expression of CARP XI in normal tissues ...... 27 2.5.2 Expression of CARP XI in pathological conditions ...... 28 2.5.3 Catalytic activity and inhibition of mutant CARP XI ...... 29

3 Aims and objectives of the study ...... 30

4 Materials and methods ...... 31

4.1 Bioinformatic analysis of CARP VIII, X and XI sequences (I) ...... 31

4.2 Expression of CARP genes (I, III, and IV) ...... 31 4.2.1 Extraction of mRNA from mouse and zebrafish ...... 31 4.2.2 Synthesis of single strand cDNA and RT-qPCR...... 32 4.2.3 Immunohistochemistry of CARPs in mouse and zebrafish (I, III)...... 33 4.2.4 Bioinformatic analysis of CARP VIII (III) ...... 34 4.2.5 Bioinformatic analysis of CARP X and XI sequences (IV) ...... 34

4.3 Molecular analysis of CARPs in zebrafish (III, IV) ...... 35 4.3.1 Zebraﬁsh husbandry for the knockdown experiments (III, IV) ...... 35 4.3.2 Sequencing of zebrafish CARP genes (III, IV) ...... 36 4.3.3 Knockdown of CARP genes using morpholinos (III, IV) ...... 36 4.3.4 Analysis of CARP VIII protein and CARP mRNAs (III, IV) ...... 36 4.3.5 Live image analysis of zebraﬁsh phenotypes (III, IV) ...... 37 4.3.6 Histochemical analysis of morphant and control larvae (III, IV) ...... 37 4.3.7 Assay to detect apoptotic cell death in morphant fish (III, IV) ...... 38 4.3.8 Electron microscope analysis (III) ...... 38 4.3.9 Cloning of human CA10 and CA11 genes in pcDNA3.1 ...... 39 4.3.10 Synthesis of mRNA and rescue of ca10a and ca10b morphants ...... 39

5 Results ...... 40

5.1 Distribution and expression of CARP VIII, X and XI sequences...... 40 5.1.1 Distribution and evolutionary analysis of CARP sequences (I) ...... 40 5.1.2 Expression analysis of CARPs in mouse tissues (I) ...... 41 5.1.3 Comparative analysis of CARP VIII sequence (II, III) ...... 42 5.1.4 Bioinformatic analysis of zebrafish CARPs and their genes (III, IV) ...... 43 5.1.5 Features of secretory proteins in CARPX-like sequences ...... 43 5.1.6 Expression analysis of CARP genes in zebrafish (III, IV) ...... 44 5.1.7 ca8 knockdown leads to developmental defects in zebrafish (III) ...... 45 5.1.8 Suppression of ca10a and ca10b genes leads to phenotypic defects (IV) .. 46 5.1.9 Morphological changes and apoptosis in morphant fish (III, IV)...... 47 5.1.10 Abnormal swim pattern in ca8, ca10a and ca10b morphants (III, IV) ...... 47

6 Discussion ...... 48

4 6.1 Bioinformatic and molecular analysis of CARP sequences ...... 48 6.1.1 The CARPs are widely distributed across species (I, IV) ...... 48 6.1.2 CARPs are mainly expressed in the CNS (I) ...... 50 6.1.3 Sequence and structural analysis of CARP VIII (II) ...... 51 6.1.4 Zebrafish CARP VIII and ITPR1 sequences are coevolved (III)...... 52 6.1.5 The ca8 morphants displayed abnormal swim pattern (III) ...... 52 6.1.6 Zebrafish ca10a and ca10b genes are widely expressed (IV) ...... 54 6.1.7 Ataxia and apoptosis in ca10a and ca10b morphants (IV) ...... 56

7 Summary and future directions ...... 57

Acknowledgements ...... 59

References...... 64

Original communications ...... 70

List of original communications

This thesis is based on the following original communications, which are referred to in the text by their Roman numerals (I-IV)

I. Aspatwar A, Tolvanen ME, Parkkila S. 2010. Phylogeny and expression of carbonic anhydrase-related proteins. BMC Mol Biol. 11:25.

II. Aspatwar A, Tolvanen M, Ortutay C, Parkkila S. 2010. Carbonic anhydrase related protein VIII and its role in neurodegeneration and cancer. Curr Pharm Des. 16:3264-76.

III. Aspatwar A, Tolvanen MEE, Jokitalo E, Parikka M, Ortutay C, Rämet M, Vihinen M, Parkkila S. 2013. Abnormal cerebellar development and ataxia in CARP VIII morphant zebrafish. Hum Mol Genet. 22:417-32.

IV. Aspatwar A, Tolvanen MEE, Barker H, Ortutay C, Pan P, Kuuslahti M, Parikka M, Rämet M, Parkkila S. 2014. Abnormal embryonic development and movement pattern in ca10a and ca10b morphant zebrafish. Submitted.

The original publications have been reproduced with the permission of the copyright holders.

6 Abbreviations

aa amino acid bp base pair BSA bovine serum albumin BLAST Basic Local Alignment Search Tool BLAT Blast-Like Alignment Tool BMD bone mineral density CA carbonic anhydrase CA8 carbonic anhydrase 8 gene (human) CA10 carbonic anhydrase 10 gene (human) CA11 carbonic anhydrase 11 gene (human) ca8 carbonic anhydrase 8 gene (zebrafish) ca10a carbonic anhydrase 10a gene (zebrafish) ca10b carbonic anhydrase 10b gene (zebrafish) cah-1 carbonic anhydrase 1 gene (Cenorhabditis elegans) cah-2 carbonic anhydrase 2 gene (Cenorhabditis elegans) Car8 carbonic anhydrase 8 gene (mouse) Car10 carbonic anhydrase 10 gene (mouse) Car11 carbonic anhydrase 11 gene (mouse) CARP VIII carbonic anhydrase related protein VIII CARP X carbonic anhydrase related protein X CARP XI carbonic anhydrase related protein XI CARP-A carbonic anhydrase related proteins (fruit fly) CARP-B carbonic anhydrase related proteins (fruit fly) cDNA complementary deoxyribonucleic acid CNS central nervous system DAB 3, 3’-diaminobenzidine tetrahydrochloride dpf day post fertilized EDTA ethylene-diamine-tetra-acetic acid EST expressed sequence tags GPI glycosylphoshpatidylinositol hpf hours post fertilized HRP horseradish peroxidase IP3 inositol trisphosphate-3 ITPR1 inositol trisphosphate receptor1 protein itpr 1a inositol trisphosphate receptor 1a gene (zebrafish) itpr 1b inositol trisphosphate receptor 1b gene (zebrafish)

7 kb kilo base MJD Machado-Joseph Disease kDa kilo Dalton LB Luria-Bertani mRNA messenger ribonucleic acid MEGA molecular and evolutionary genetic analysis MO Morpholino oligonucleotide MSA multiple sequence alignment NB Northern blot PBS phosphate buffered saline PCs Purkinje cells PCR polymerase chain reaction p53 gene for Tumor protein p53 RC random control RT-PCR reverse transcriptase-polymerase chain reaction RT-qPCR real-time quantitative polymerase chain reaction SNP single nucleotide polymorphism SCA spinocerebellar ataxia SDS sodium dodecyl sulfate TUNEL terminal deoxynucleotidyl transferase dUTP nick end labeling UCSC University of California Santa Cruz WB Western blotting wdl waddles wd waddler WT wild type

8 Abstract

The α-carbonic anhydrases (CAs) are zinc containing metalloenzymes whose primary role is to catalyze the reversible hydration of carbon dioxide to bicarbonate and a hydrogen ion. The α-CAs are found abundantly in nature and participate in many biologically important processes apart from reversible hydration of carbon dioxide. Among the α-CA gene family, there are three homologues that have no catalytic activity due to lack of one or more of the three functionally important histidine residues and are named as carbonic anhydrase related proteins (CARPs) VIII, X and XI. Interestingly, the CARPs VIII, X, and XI are predominantly expressed in all parts of the brain in humans and mice. However, the precise physiological roles of these proteins are poorly understood. The main aim of the present work was to perform a systematic analysis of CARPs in order to elucidate their phylogenesis, distribution, and functional role. Among the three CARPs, CARP VIII was first to be reported from the mouse complementary DNA (cDNA) library. Mutations in CA8 gene have been linked to ataxia and mental retardation in humans and mice. CARP VIII interacts with inositol tris-phosphate receptor1 (ITPR1) and might be involved in intracellular Ca2+ signaling. Some studies have suggested that CARP X is expressed in mouse and human brain. CARP XI is also predominantly expressed in the central nervous system (CNS) and upregulation of CARP XI has been reported in some cancers. Database searches and phylogenetic analyses showed a nearly universal distribution for CARP X-like sequences in vertebrates and invertebrates, whereas CARP VIII was limited to chordates and a few invertebrates (deuterostomes). In contrast, CARP XI is limited to vertebrates, and was found to be missing in fish and bird lineages. Surprisingly, many fish species exhibited independent duplication of CARP X sequences. Expression of CARP proteins and their messenger ribonucleic acids (mRNAs) were studied using immunohistochemistry and real-time quantitative polymerase chain reaction (RT-qPCR), respectively. At the protein level CARP VIII demonstrated very strong expression in the cerebellum and cerebrum; moderate expression in the lung, liver, salivary gland, and stomach; and low expression in the colon and kidney. CARP X was expressed in the lung and a weak signal was observed in the kidney. Low levels of CARP XI expression was seen in the cerebellum, cerebrum, stomach, kidney, liver, and small intestine, and weak staining was also seen in the colon. RT-qPCR analyses confirmed the results of the immunohistochemical staining, showing wide distribution of Car8 and Car11 mRNAs in different tissues, whereas the expression of Car10 mRNA was restricted to the frontal cortex, parietal cortex, cerebellum, midbrain, and eye. CARPs are widely distributed with a very high

9 sequence similarity among the organisms analyzed in the study. Distribution of CARPs and Car mRNAs suggests that these proteins may play role in brain development and motor coordination function in the cerebellum, and that they might also play some physiological role in other tissues. The comparative analysis of human CARP VIII structure with catalytically active human CA II indicated that the surface of the CARP VIII protein molecule is highly conserved. Whether or not the conserved surface of CARP VIII is involved in interaction with ITPR1 remains to be studied in the future. The structural basis of the misfolding of the S100P mutant protein associated with ataxia can be suggested based on the crystal structure of the wild-type CARP VIII. Substitution of Ser to Pro will cause a rigid bend in the peptide backbone, which might prevent the β6 and/or the β6/β7 loop from assuming the conformations they have in the wild-type protein. Having a shorter, more constrained loop between strands β5 and β6 may amplify the problem in folding of the S100P mutant protein even further. The presence of CARP orthologs in zebrafish led us to investigate their role by knocking down the CARP genes using antisense morpholino oligonucleotides (MOs). The expression analysis of CARP VIII in zebrafish showed that CARP VIII is expressed in the cerebellar Purkinje cells (PCs) similar to the distribution in humans. Bioinformatic analysis revealed that the CARP VIII and ITPR1 sequences are coevolved. Knockdown of CA8 gene in 0-5 day post fertilized (dpf) zebrafish embryos led to abnormal cerebellar development and ataxia in 5 dpf ca8 morphants similar to human patients with a mutation in the CA8 gene. Similarly, developmental expression of ca10a and ca10b genes suggested that these genes are required for the embryonic development in 1-5 dpf embryos. Expression studies on ca10a and ca10b genes in adult tissues suggested that these genes are highly expressed in the brain, similar to the expression pattern in human and mouse. Bioinformatic analysis revealed conserved signal peptides, N-glycosylation sites and potential disulfides, suggesting that CARP Xa and Xb proteins are secretory and potentially dimeric. Knockdown of these genes using antisense MOs led to phenotypic abnormalities in 5 dpf morphant zebrafish with gross morphological changes in the brain and ataxic movement. Our studies introduced novel zebrafish models to further investigate the mechanisms of CARP functions in vertebrates.

10 1 Introduction

The α-carbonic anhydrases (α-CAs) are Zn2+ containing enzymes that catalyze the reversible hydration of CO2 (Sly and Hu, 1995). There are 17 α-CAs in vertebrates, of which 14 isozymes (CA I, II, III, IV, VA, VB, VI, VII, IX, XII, XIII, XIV, XV, and XVII) are catalytically active (Tolvanen et al., 2013). Three of the isoforms, namely, CARPs VIII, X, and XI, represent inactive isoforms due to lack of one or more of the three His residues required for binding to the Zn2+ atom essential for CA activity (Hewett-Emmett and Tashian, 1996). Among the inactive CA isoforms, the CARP VIII gene was the first to be reported from the mouse brain cDNA library (Kato, 1990a). The catalytic inactivity of CARP VIII is due to the lack of one of the three His residues essential for zinc binding. CARP VIII has been well studied compared to CARP X and CARP XI (Lakkis et al., 1997a; Taniuchi et al., 2002a; Taniuchi et al., 2002b). CARP VIII is primarily expressed in cerebellar PCs, and its expression has also been reported in several other normal and cancer tissues (Akisawa et al., 2003; Kato, 1990b; Miyaji et al., 2003; Taniuchi et al., 2002b). CARP VIII interacts with ITPR1, a Ca2+ channel protein (Hirota et al., 2003). The crystal structure of human CARP VIII has been resolved and the structural basis of its inactivity has been reported (Picaud et al., 2009). CARP VIII protein deficit is also associated with ataxia and mental retardation both in mouse and human (Jiao et al., 2005; Kaya et al., 2011; Turkmen et al., 2009). Similar to CARP VIII, both CARP X and CARP XI are catalytically inactive owing to the absence of two and three histidine residues, respectively, which are required for CA activity. Unlike CARP VIII, so far, very few studies have been reported on CARP X and CARP XI. Most of the studies have been mainly related to their expression in human and mouse (Taniuchi et al., 2002a; Taniuchi et al., 2002b). Similar to CARP VIII, both CARP X and CARP XI are predominantly expressed in the CNS and CARP XI is also upregulated in gastrointestinal tumors (Morimoto et al., 2005). Apart from the few expression related studies very little is known about CARP X and CARP XI proteins. The purpose of the present work is to study the distribution of CARP sequences across the species and to study the expression pattern of CARPs at both gene and protein levels in mouse tissues. In addition, analysis of databases revealed the presence of CARP orthologs in zebrafish, and therefore we used zebrafish as a vertebrate model to study the expression pattern during embryonic development and in adult tissues. Furthermore, we knocked down the CARP genes in zebrafish using antisense morpholinos to study their role in the zebrafish model.

2 Review of the literature

2.1 Carbonic anhydrases

2.1.1 Historical aspects of carbonic anhydrase isozymes

The carbonic anhydrases (CAs, EC 4.2.1.1) are ubiquitous metalloenzymes and their basic function is to catalyze the reversible hydration of CO2 in the reaction: CO2+H2O  H++HCO3-, which is important for many biological processes, such as photosynthesis, respiration, renal tubular acidification, and bone resorption (Sly and Hu, 1995). The CAs have been found in almost every living organism, and are encoded by five distinct evolutionarily unrelated gene families denoted as α, β, γ, δ and ζ CAs (Hewett-Emmett and Tashian, 1996). These gene families do not share any structural similarity between each other, and indeed, they are good examples of convergent evolution of catalytic function (Hewett-Emmett and Tashian, 1996). The CAs of various animal species belong to the α-CA gene family and contain Zn2+ in the active site. In mammals, only 13 enzymatically active α-CAs have been identified, (CA I, II, III, IV, VA, VB, VI, VII, IX, XII, XIII, XIV, and XV). The 13 active CA isozymes have different subcellular localizations such that CAs I, II, III, VII, and XIII are all cytosolic, CAs IV, IX, XII, XIV, and XV are all membrane- associated, CAs VA and VB are mitochondrial, and CA VI is a secreted protein (Hewett-Emmett, 2000; Hewett-Emmett and Tashian, 1996; Sly and Hu, 1995). In addition to the active CAs, there are CA isoforms which have sequence and structural similarity with active CAs but do not possess any CA catalytic activity. In mammals, the catalytically inactive CA isoforms occur either independently, and are named as carbonic anhydrase related proteins (CARPs), or they exist as domains of other proteins. The catalytic inactivity of these CA isoforms is due to the absence of one or more of the three histidine residues required for the classical enzymatic activity (Sly and Hu, 1995; Tashian et al., 2000).

2.2 Carbonic anhydrase related proteins

The catalytically inactive CAs and CA domains of other proteins lacking CA activity were discovered in the 1990s (Kato, 1990b; Maa et al., 1990). There are three CARPs which exist in all vertebrates and two CARPs which are part of other proteins occurring as domains of protein tyrosine phosphate receptor (PTPR) ζ or β and γ

12 (Ohradanova et al., 2007). In addition to the presence of CARPs and CARP domains in the vertebrates, the D8 transmembrane protein in the vaccinia virus contains an N-terminal CA domain (Maa et al., 1990) (Table 1).

Table 1. Molecular properties of CARPs CARPs Accession Noa. Amino acids (aa) MW (kDa) Chromosomal localization VIIIc AK090655 290 33 8q11-q12 Xc AF288385 328 37.6 17q24 XIc AF067662 328 36.2 19q13.3 PTPRζc NP_002842 280b - 7q31-33 PTPRγc NP_002832 266b - 3pl4.2-p2l CA in D8d J05190 236b 32 - aThe accession numbers represent the longest cDNA sequences deposited in the GenBank. bThe length of CA domain. cHuman and dVaccinia virus CARPs (Ohradanova et al., 2007; Supuran, 2004).

The CARPs VIII, X, and XI were discovered based on the sequence homology with the catalytic CA isozymes (Tashian et al., 2000). The Car8 gene was first reported by Kato from the mouse brain cDNA library (Kato, 1990a). The presence of CA10 was mentioned by Hewett and Tashian based on the expressed sequence tags (ESTs) which showed very high homology to CA isozymes, and the predicted protein was devoid of the histidine residues essential for CA catalytic activity (Hewett-Emmett and Tashian, 1996). CARP XI was accidentally found in 1998 and reported as CARP-2 (Bellingham et al., 1998). The sequences of CARP VIII, X and XI are highly conserved and represent a sub-class of the α-CA gene family. Importantly, they lack one or more of the three histidine residues which are essential for coordinating the zinc atom at the active site and therefore, they lack the CA enzymatic activity (Hewett-Emmett and Tashian, 1996). In CARP VIII, one of the His is replaced by Arg and in CARP X two of the His residues are replaced by Arg and Gln. In CARP XI, all the three important histidines are replaced by Arg, Leu, and Gln (Fig 1) (Bellingham et al., 1998; Fujikawa-Adachi et al., 1999; Lovejoy et al., 1998; Okamoto et al., 2001; Skaggs et al., 1993). Based on the phylogenetic analysis of the human α-CAs, the fourteen CA isozymes are grouped into extracellular and intracellular CAs (Okamoto et al., 2001). The phylogenetic analysis indicated that three CARPs are grouped together forming a single divergent branch that shares the same branch as intracellular CA isozymes, suggesting that CARPs are intracellular. The protein tyrosine phosphatases (PTPs) regulate cell proliferation, differentiation, communication and adhesion by dephosphorylating tyrosine residues of proteins (Andersen et al., 2004). The PTPRs are integral cell surface proteins which have PTP activity. There are eight subfamilies (R1/R6, R2A, R2B, R3, R4, R5, R7 and R8) of PTPRs based on the structure of their extracellular domains (Andersen et al., 2004) ). The PTPRs ζ and γ, which belong to the R5 subfamily, are known to contain N-terminal CARP domains and lack enzymatic activity due to the absence of two of the three His residues required for binding to the zinc atom (Barnea et al., 1993; Gavrieli et al., 1992; Levy et al., 1993). The CARP

13 domains of PTPRs ζ and γ genes are made up of seven exons and six introns which are similar to the introns of mammalian CA1,2,3,5 and 7 genes (Kastury et al., 1996). The CARP domains of these receptor proteins have close to 50% aa similarity with mammalian CAs (Barnea et al., 1993; Levy et al., 1993). In PTPR ζ two of the three His residues required for the co-ordination of Zn2+ at the active site are replaced by Thr at position His-94 and Gln at position His-119 (Fig 1). Similarly, in PTPRs γ two His obligatory for binding to the Zn2+ at the active site are replaced by Glu at position His-94 and Gln at position His-119 (Fig 1) making the proteins catalytically inactive (Ohradanova et al., 2007). The PTPR ζ is present on the surface of glial cells where it interacts with contactin, a cell recognition molecule, via CA domain and thus plays a role in neuronal cell adhesion and neurite outgrowth (Peles et al., 1995). The PTPR γ protein is expressed in several different tissues and is associated with many diseases including renal and lung cancers in humans and motor coordination function in mice (LaForgia et al., 1991; Lamprianou et al., 2006).

Figure 1. Alignment of aa residues 64–128 (CA II numbering, relevant for the catalytic activity) of hCARPs, hCAs I–XIV, hRPTPβ/ζ, γ and vaccinia virus CA. The Zn2+ binding His of CAs and corresponding residues in the CARPs are in red and indicated with arrow. Modified from (Ohradanova et al., 2007).

The presence of the CA domain in the D8 transmembrane protein at the N- terminal region was reported in 1990 (Maa et al., 1990). The N-terminal domain of D8 protein contains 236 aa residues which are homologous to the catalytic domain of the mammalian CAs. In CA domain of D8 protein the His residues at positions 69 and 92 which correspond to positions 96 and 119 in CAII, are replaced by Tyr and Asn, respectively, making it catalytically inactive (Fig 1) (Ohradanova et al., 2007). Phylogenetic analysis revealed that the viral CA domain is related to CARP X and XI, indicating that the vaccinia virus might have obtained the CA domain from humans via horizontal transfer of the gene (Ohradanova et al., 2007). The molecular weight (MW) of the CA domain of D8 protein was 32 kDa (kilo Dalton) with a pI of ∼ 8.7. The mutated protein with a replacement of Tyr and Asn (vaccCA

14 N92H/Y69H) with His residues showing catalytic activity similar to the catalytic activity of human CA VA and CA XII (Ohradanova et al., 2007). Apart from the CARPs and CARP domains found in vertebrate species, the invertebrates were found to contain CARP X like sequences, unlike CARP VIII orthologs which are found only in deuterostome invertebrates, with the exception of single protostome species (Miyamoto, 2012). Many protostome species, such as C. elegans, a nematode and insects like D. melanogaster contain more than one copy of CARP X like sequences (Ortutay et al., 2010). Interestingly, these CAX-like sequences are expressed in the neuronal cells of insects and nematodes (Tolvanen et al unpublished result).

2.3 General properties of CARP VIII

CARP VIII was discovered from mouse cDNA clones with a specific expression pattern in the brain (Kato, 1990a). The nucleotide sequence analysis of these clones exhibited a strong homology with α-CAs (Kato, 1990b). CARP VIII showed replacement of His and Glu residues by Arg and Gln at positions 124 and 122. His 124 is one of the three histidines essential for the co-ordination of the Zn2+ at the active site and Gln residue at position 122 forms a hydrogen bond network to the zinc bound solvent molecule (Tashian, 1989). In addition to high homology with CAs, CARP VIII had a Glu stretch at the N-terminal end and it was only expressed in cerebellar PCs (Kato, 1990b). CARP VIII is well characterized inactive isoform and many studies have been done related to its expression (Hirota et al., 2003; Lakkis et al., 1997a; Taniuchi et al., 2002a; Taniuchi et al., 2002b) and the role of CA8 in lung and colon cancer (Akisawa et al., 2003; Lu et al., 2004; Miyaji E et al., 2003; Miyaji et al., 2003; Nishikata et al., 2007). In the past, the association of CARP VIII mutation with ataxia has been reported in mouse (Jiao et al., 2005). Recent studies revealed a mutation in the CA8 gene which leads to ataxia in humans (Kaya et al., 2011; Turkmen et al., 2009). The crystal structure of CARP VIII has been resolved and its structural basis of inactivity has also been studied (Picaud et al., 2009). In addition to the above studies CARP VIII has been implicated in several other diseases (Bataller et al., 2004; Miyaji et al., 2003; Mori et al., 2009).

2.3.1 Expression of CARP VIII in animal models

The expression of the Car8 gene was originally studied in mouse using Northern blot (NB) and in-situ hybridization (Kato, 1990a). The mRNA exhibited a unique distribution in neuronal PCs of the cerebellum (Kato, 1990b; Lakkis et al., 1997b). Subsequently, several studies were carried out related to CARP VIII gene expression in both mouse and human tissues. In-situ hybridization suggested that the Car8 gene

15 is expressed at 9.5 days of gestation in several organs in the developing mouse. The high level of expression was seen between 10.5 to 12.5 days of development in the brain, liver, lung, heart, gut, thymus and epithelium covering the head and oronasal cavity (Lakkis et al., 1997a). In-situ hybridization and immunohistochemistry revealed the presence of CARP VIII mRNA and its protein product in the PCs of 9-day-old mouse (Nogradi et al., 1997). Reverse transcriptase (RT) PCR and NB of whole mouse embryos at 7, 11, 15 and 17 gestational days showed strong signals for the Car8 gene in 11, 15 and 17 days of gestation, supporting the earlier findings that the Car8 gene appears after 7 days during development of the embryo (Lakkis et al., 1997a; Taniuchi et al., 2002b). In adult mouse tissues, Car 8 mRNA was predominantly found in the brain and the expression was also seen in the lung, liver, heart, skeletal muscle and kidney (Taniuchi et al., 2002b). Western blot of CARP VIII protein in a panel of mouse tissues revealed that it is predominantly expressed in the cerebellum similar to the expression of Car8 mRNA. In addition, low expression was also seen in the cerebrum, olfactory bulb, olfactory epithelium, vomeronasal organ, lung, submandibular gland, liver, adrenal gland, stomach, small intestine and large intestine (Hirota et al., 2003). However, there was no signal for the presence of CARP VIII protein in the heart, thymus, spleen, pancreas, ovary, uterus, testis or muscle (Hirota et al., 2003). The microarray analysis of single rod bipolar cells indicated the enrichment of the Car8 gene in these cells, and the expression of Car8 gene was subsequently confirmed by in-situ hybridization using RNA probes (Kim et al., 2008). Recently, the localization pattern of CARP VIII was studied in retinal sections of mice that were heterozygous or homozygous for a mutation in waddles (wdl) gene (Puthussery et al., 2011). Immunohistochemistry showed CARP VIII protein in the inner retina of the heterozygous mouse, but the retina of the homozygous mouse for wdl mutation exhibited no signal. The pattern of CARP VIII localization was similar in the retina of rat and macaque, suggesting conservation of CARP VIII expression in mammals (Puthussery et al., 2011).

2.3.2 Expression of CARP VIII in humans and human cell line

The polymerase chain reaction (PCR) amplification of human CA8 gene from cDNA libraries of human tissues revealed the presence of CA8 in placenta, salivary gland and testis (Skaggs et al., 1993). The regional and cellular distribution of CARP VIII protein was analyzed in the sections of adult and fetal brains at five different gestational periods (Taniuchi et al., 2002a). In the fetal brain the signal for CARP VIII protein was seen in neuroprogenitor cells and sub-ventricular zone at 84 days of gestation, while the distribution was more widespread in the adult brain (Taniuchi et al., 2002a). The expression analysis using microarray suggested that the CA8 gene is predominantly expressed in the cerebellum, and high levels of CA8 gene was also seen in several other tissues (Kilpinen et al., 2008; Wu et al., 2009).

16 The gene expression profile of the human neuroblastoma cell line exhibited a 9 fold increase in the expression of CA8 gene when mutant ataxin 3 was present in the cells compared to the cells containing wild type (WT) ataxin 3 (Wen et al., 2003). Recent studies on the distribution of CARP VIII protein displayed a significant increase of CARP VIII protein in the neuroblastoma cells (SK-N-SH) expressing mutant ataxin 3 protein (Hsieh et al., 2013). Immunochemical analysis showed that the CARP VIII protein was evenly distributed in the perinuclear and cytoplasmic regions. Interestingly, ITPR1, which is expressed in the endoplasmic reticulum co- localizes with CARP VIII in the perinuclear region (Hirota et al., 2003). Studies done earlier suggested that the mutant ataxin 3 disrupts Ca2+ signaling in the neurons by interacting with ITPR1 on the surface of the endoplasmic reticulum (Chen et al., 2008). Studies in the cultured cells also suggested that the binding of mutant ataxin 3 to ITPR1 increases its sensitivity to inositol tris-phosphate-3 (IP3), leading to alteration in Ca2+ signaling. Therefore, it appears that the presence of mutant ataxin 3 in the neuronal cells changes the localization of CARP VIII, along with its increased expression and leads to disruption of ITPR1-mediated calcium signaling in the neurons (Hsieh et al., 2013).

2.3.3 Crystal structure of CARP VIII and its interaction with ITPR1

The crystal structure of human CARP VIII (hCARP VIII) has been determined at 1.6 Å resolution (2W2J in PDB) (Picaud et al., 2009). The 3-dimentional structure of hCARP VIII (Fig 2) has a Glu stretch at the N-terminal end from aa 24 to 36 (E loop). CARP VIII has the central core domain (aa 37 to aa 290) which is similar to the mammalian CA enzymes. The core domain consists of 10-stranded central β- sheets surrounded by several α-helices and β-strands. The crystal structure of CARP VIII has similarity with cytosolic human CA XIII (hCA XIII) (41% sequence identity) and has several differences in two loop regions (Fig 3 II). In hCARP VIII, the E loop protrudes into the exterior and packs against the α3-β15 loop, which incorporates a five-residue insertion compared with other isozymes. Compared with hCA II the hCARP VIII cavity is spatially narrower due to replacement of H94, which is essential for binding to the zinc in the active site. Other residues in the active site essential for CA enzymatic activity in CAII, such as T200, V121, and V143 in CAII were substituted by I224, I143 and I165, respectively. The replaced aa are bulkier, reduced the pocket size in hCARP VIII, and obstruct the binding of CO2 in the active site. The crystal structure showed that the central core domain allows the CARP VIII protein to adopt the classic CA fold but the replacement of aa residues, which play a key role in CA enzymatic activity, rendered the CARP VIII catalytically inactive. Interestingly, replacement of R116, E114, and I224 with H117, Q115, and T225 makes the CA VIII enzymatically active (Elleby et al., 2000; Sjoblom et al., 1996). However the sequence and structural similarity of hCARP VIII with active CAs suggest that over the course of evolution CARPs have

17 gained new functions possibly to control the function of other proteins through the protein-protein interactions.

Figure 2. Crystal structure of hCARP VIII. (A) Ribbon diagram exhibiting the secondary structure elements: α-helices (red) and β-strands (yellow). (B) Cα-superposition of hCARP VIII (blue) and hCA XIII structures (cyan, PDB code 3DA2). The two loop regions that are unique in hCARP VIII are shown in thick ribbons. Source: (Picaud et al., 2009).

It has been reported that ITPR1, which interacts with CARP VIII, has an electropositive IP3 binding site (Bosanac et al., 2002; Hirota et al., 2003). The unique electronegative surface in CARP VIII could form a charge-complementary binding site for ITPR1 and might be involved in the release of Ca2+ from endoplasmic reticulum (Jiao et al., 2005). ITPR1, which is expressed in the PCs of cerebellum similar to CARP VIII, has been identified as a binding partner for CARP VIII (Hirota et al., 2003). The deletion mutation analysis of CARP VIII revealed that the aa region from 45 to 291 interacts with the regulatory domain of ITPR1, which is located between aa 1387-1647 (Fig 3). It is suggested that CARP VIII reduces the sensitivity of ITPR1 to IP3 and thereby controlled release of Ca2+ in the PCs. This finding suggests that the co-expression of CARP VIII with ITPR1 in PCs has an inhibitory effect on IP3 binding (Hirota et al., 2003).

Figure 3: Schematic presentation of ITPR1 presenting the binding regions for calmodulin, IP3 at N- terminal end and a channel pore at C-terminal region. The regulatory region of ITPR1 shows the region of aa 1387-1647 which interacts with CARP VIII protein. Modified from (Mikoshiba, 2007).

18 2.3.4 Role of Car8 gene in gait disorder in mouse

The association of Car8 gene mutation with ataxia was first reported from studies on wdl mice (Jiao et al., 2005). The mice which had a spontaneously occurring mutation in Car8 gene were characterized by wobbly side-to-side ataxic movement when the mice reached two weeks of age, and the disorder persisted throughout their life span. Further studies on these mice revealed a 19 base pair (bp) deletion in exon 8 of the Car8 gene responsible for waddled phenotype. The Car8 gene was located in the genetic region of wdl and the deletion of Car8 was the only defect found among the genes and ESTs within the wdl locus of wdl mice. Similarly, the wdl mice had no detectable CARP VIII protein. The Car8 (wdl) mutation was autosomal recessive and it was located close to the waddler (wd) locus on mouse chromosome 4 (Yoon 1959). The sequence analysis of the affected gene showed a deletion of 19 bp, in the exon 8 of Car8 gene and caused a shifting of the reading frame. The shifting of the reading frame inserted a stop codon in the gene which eliminated 50 aa in the protein leading to a non-functional protein (Jiao et al., 2005). Further, structural level studies in these mice exhibited functional abnormalities of excitatory synapses of PCs. The functional abnormalities of excitatory synapses of PCs might be responsible for motor coordination defects in these mice (Hirasawa et al., 2007). These studies suggest the importance of CARP VIII in the development and/or maintenance of the proper morphology and function of PC synapses (Hirasawa et al., 2007).

2.3.5 Association of CA8 gene with ataxia in humans

The involvement of CA8 gene in human neurodevelopmental disorder was suggested based on the studies in members of Iraqi and Saudi Arabian families (Kaya et al., 2011; Turkmen et al., 2009). In the first study, the genetic analysis of members of the Iraqi family showed a homozygous missense mutation, where serine 100 was replaced by proline (S100P) in the CARP VIII protein (Turkmen et al., 2009). The affected members exhibited a mild mental retardation and congenital ataxia accompanied by a quadrupedal gait (Fig 4) (Turkmen et al., 2009). In-vitro studies suggest that the change produced by the S100P change leads to proteasome- mediated elimination of CARP VIII protein due to misfolding. This is a common mechanism in cases of loss of function mutations which are associated with various human genetic diseases (Lukacs and Verkman, 2012; Wang and Moult, 2001). The affected members of the Iraqi family had a phenotype similar to wdl mice (Jiao et al., 2005). In this study, the clinical features of the patients included cerebellar ataxia, dysarthria, mild mental retardation and tremor (Turkmen et al., 2009).

Figure 4. Members of the Iraqi family with a quadrupedal gait. With permission (Turkmen et al., 2009).

In the second study, all the seven affected members of Saudi Arabian family had cerebellar ataxia, mental retardation, and disequilibrium syndrome type 3 (Kaya et al., 2011). Sequencing of exons revealed a novel homozygous variation (484G>A) in exon 4 of CA8 gene of all the affected members and the parents of the affected members showed heterozygous variation (484G>A) in exon 4 of CA8 gene. Homozygosity for the variation cosegregated with the disease phenotype and the substitution was not present in 200 unrelated healthy controls of the same ethnic origin, indicating that the G162R (484G>A) variation in CARP VIII protein is very likely to be pathogenic. The clinical features in the patients included variable cerebellar ataxia and mild cognitive impairment and interestingly none of the patients had a quadrupedal gait. The affected patients showed loss in the cerebellar volume (Fig 5) and ill-defined peritrigonal white matter abnormalities along with hypometabolic cerebellar hemispheres, temporal lobes, and mesial cortex. The common clinical features between affected members of the present study and the members of Iraqi family were mild cognitive impairment, variable cerebellar ataxia, absence of seizures, and lack of dysmorphism. In this study the absence of a quadrupedal gait could probably be associated with the environmental factors rather than genuine phenotypic change due to the disease (Kaya et al., 2011).

Figure 5. The cerebellar volumes of Saudi Arabian patients. The progressive cerebellar loss (arrows) in patient 1 between the sagittal T1 (A and C) and axial flair images (B and D) at 4 (A, B) and 8 years of age (C, D), respectively. Sagittal T1 images indicate reduction in cerebellar volume more prominent in patient 3 (E) than in patient 2 (F). Modified with permission (Kaya et al., 2011).

The phenotypic changes observed in mice and humans could be due to altered Ca2+ signaling because of the mutation in CARP VIII gene which increased the sensitivity of ITPR1 to IP3 (Jiao et al., 2005; Kaya et al., 2011; Turkmen et al., 2009). The release of intracellular Ca2+ ions from endoplasmic reticulum is responsible for several cellular activities which include: synaptic recycling, proliferation, fertilization, learning and memory, long-term potentiation, depression, apoptosis, contraction, metabolism, and the control of other signaling systems. In fact, the cerebellar gene expression profile of wdl mouse revealed that many genes involved in synaptogenesis, synaptic vesicle formation and transport, cellular proliferation and differentiation, and signal transduction were dysregulated (Yan et al., 2007). These findings suggest that ultrastructural abnormalities of specific neuronal elements may be present in wdl cerebellar cortex. Similarly, dysregulation of many genes in wdl mouse suggests that the Car8 gene may be playing a role in the cerebellar cortex (Yan et al., 2007).

2.3.6 Role of CARP VIII in cancer and other diseases

The potential role of CARP VIII was investigated in human colorectal epithelial carcinomas by immunohistochemistry (Miyaji et al., 2003). Expression of CARP VIII and Ki-67, a marker for cell proliferation, was studied in 60 human adenocarcinomas and 13 human adenoma samples. The expression of CARP VIII

21 was found in 78% of colorectal carcinomas, whereas only 5% of the 73 normal colon samples were positive for CARP VIII. The signal for CARP VIII was observed more frequently at the tumor invasion front compared with Ki-67 antigen. CARP VIII staining for colorectal adenoma was significantly lower in comparison with adenocarcinomas (Miyaji et al., 2003). The selective expression of CARP VIII at the tumor invasion front suggests that it plays a role in invasion of colorectal cancer. Further the role of CARP VIII was explored using colon cancer cell lines (LoVo- CA8) (Nishikata et al., 2007). The studies on LoVo-CA8 and the control cell line (LoVo-pCIneo) suggested that the LoVo-CA8 cell line expressed high levels of CA8 mRNA and the corresponding CARP VIII protein. The LoVo-CA8 cell line showed higher cell proliferation and cell invasion abilities compared to the parental LoVo and LoVo-pCIneo cells . The tumor growth in mice transplanted with LoVo-CA8 cells was higher than the parental LoVo cells. The knockdown of CA8 using small interfering RNA in another cell line (HCT116), expressing high level of endogenous CARP VIII, efficiently inhibited cell proliferation and colony formation. This result also suggested that the CARP VIII plays a role in colon cancer. Comparative expression studies of CARP VIII in mouse and human tissues exhibited a very faint signal for CA8 in adult human lung compared with the signal for Car8 in mouse lung (Akisawa et al., 2003). However, the expression analysis in human fetal lung indicated a very strong signal for CA8 both at protein and mRNA level. Further analysis of expression by immunohistochemistry showed a signal for CARP VIII in non-tumorous lung tissues in adults. The signals for CARP VIII protein were restricted to bronchial ciliated cells, chondrocytes of bronchial cartilage, and smooth muscle cells. However, developing pulmonary epithelium showed abundant CARP VIII. This pattern of CARP VIII presence led to the study, whether CARP VIII is an oncofetal antigen in human lungs. The immunochemical staining of pulmonary non-small cell carcinoma specimens exhibited a very strong signal for CARP VIII in 54 out of 55 cases. The staining was strong at the front of the tumor progression and a weak staining was observed in the center of the carcinomas. These findings suggested that CARP VIII is strongly expressed in highly proliferative lung cancer cells (Akisawa et al., 2003). CARP VIII expression studies were done in human lung adenocarcinoma cell line (PC-9), where the exogenous CARP VIII expression leads to intracytoplasmic vacuole formation (Ishihara et al., 2006). Similarly, signet ring cells, which are seen in several carcinomas and contain intracytoplasmic mucin were observed among CARP VIII expressing PC-9 cells compared to control PC-9 cells. Expression of CARP VIII in adenomatous hyperplasia and early-stage lung adenocarcinoma suggested an abundant presence of CARP VIII in invasive lung adenocarcinoma compared with noninvasive adenocarcinoma. Matrigel invasion assay revealed invasiveness by PC-9 cells expressing CARP VIII but not by control PC-9 cells, suggesting that CARP VIII expression in lung cancer is involved in cancer cell invasion (Ishihara et al., 2006).

22 The expression of CARP VIII was studied in gastrointestinal stromal tumors (GIST) and in gastric tissues of a patient with von Recklinghausen disease (the disease causing tumors called neurofibromas in the tissues and organs of the body) by immunohistochemistry. The expression analysis of 22 GIST samples indicated high levels of CARP VIII in 13 samples (59%) and an intense signal for CARP VIII was also seen in neural cell bodies of a patient with von Recklinghausen disease. In the same study, expression of CARP VIII was analyzed in GIST-T1 cells and the same cells transfected with CA11 expression vector (GIST-T1-CA11). There was no change in the expression level of CARP VIII in these cells, but surprisingly, there was a change in the subcellular localization of CARP VIII. The immunohistochemistry suggested localization of CARP VIII in the perinuclear region of GIST-T1 cells, whereas CARP VIII was localized in the whole cell body in GIST-T1-CA11 cells. The expression pattern of CARP VIII in these studies suggested that CARP VIII might play a role in the development of GIST and may also indirectly, or directly, interplay with CARP XI (Morimoto et al., 2005). The CARP VIII protein may also act as an autoantigen, which is associated with the pathogenesis of melanoma-associated Paraneoplastic cerebellar degeneration (PCD). Even though it is a single observation, serum of a patient with PCD and malignant melanoma had reactivity with PCs, and the autoantibody showed reactivity against CARP VIII protein (Bataller et al., 2004). Association of single nucleotide polymorphisms (SNPs) in CA8 with femoral and lumbar bone mineral density (BMD) was studied in 337 women suffering from osteoporosis. The study presented a significant correlation between CA8 SNP rs6984526 and femoral BMD, and the BMD of homozygous carriers of major (C) allele was higher compared with heterozygous carriers. Similarly, CA8 SNP also suggested a very strong association with lumbar BMD. The variations of CA8 loci could be an important determinant of osteoporosis and the CA8 gene seem to have an additional role in the process of bone resorption (Mori et al., 2009).

2.3.7 Catalytic activity and inhibition of mutant CARP VIII

The restoration of two aa residues (Arg117 to His and Glu115 to Gln) required for the Zn2+ co-ordination converts the inactive mouse CARP VIII to an active enzyme (Sjoblom et al., 1996). The enzymatic activity of the mutated CARP VIII was significantly higher in comparison with the mammalian CA III and the activity could be strongly inhibited by acetazolamide (Sjoblom et al., 1996). In fact, the same authors showed that a single mutation to restore the Zn2+ binding His residue at position 117 in place of Arg is sufficient for the catalytic activity of CARP VIII but the catalytic activity was half of the double mutated CARP VIII enzyme (Elleby et al., 2000). The replacement of Gln92 to Glu in CA II showed a slight effect on its enzymatic activity, suggesting that the replacement of Glu115 with Gln in CARP VIII might lead to an increase in enzymatic activity (Elleby et al., 2000; Kiefer, 1995;

Sjoblom et al., 1996). In the same study, the authors replaced three more residues in mouse CARP VIII, namely Ile225 to Thr and two isoleucines at positions 144 and 166 to Val. The replacement of these residues was done based on the information that the Thr at that position is not required for the catalytic activity but it has a significant effect on the catalytic activity (Behravan et al., 1991; Elleby et al., 1999). Similarly, occurrences of two Val residues, which are bulky, make the active site region too small for optimal catalytic activity (Elleby et al., 2000; Kato, 1990b). Indeed, the mouse CARP VIII with an additional three mutations revealed a significantly increased activity (Elleby et al., 2000). In a recent study, the catalytic activity of hCARP VIII was restored. The mutated CARP VIII had a very high catalytic activity with a kcat/km of 67.3% of hCA II, which is one of the highly active enzymes. The mutated CARP VIII could be effectively inhibited by acetazolamide. The catalytic activity of the mutated hCARP VIII was higher than the mutated mouse CARP VIII, although there is only a difference of three aa between the mouse and human CARP VIII (Nishimori et al., 2013).

2.4 General properties of CARP X

CARP X was discovered in 1996 based on expressed sequence tags (ESTs), which displayed significant homology with catalytically active CAs (Hewett-Emmett and Tashian, 1996). Further analysis of ESTs revealed that these sequences lacked two of the three His essential for binding to a Zn2+ and were named as CA10 (Hewett- Emmett and Tashian, 1996). Later on, Kleiderlein and coworkers found a cDNA homologue showing 61% identity (accession No. AF064854) with CA11 sequence while screening human cDNA libraries for the CCG repeats (Kleiderlein et al., 1998). Subsequently, a 2720 bp full length cDNA clone was obtained encoding 328 aa protein with a predicated molecular mass of 37.6 kDa. The aa sequence deduced from 2720 bp cDNA showed 25-57% similarity with catalytically active CAs and exhibited highest identity with CARP XI (Okamoto et al., 2001). The CA10 sequence is known to contain seven CCG repeats at the 5’-untranslated region followed by two CCG repeats at 16 bp downstream region and the polymorphism of its repeat number has been reported in normal humans (Kleiderlein et al., 1998).

2.4.1 Expression of CARP X in human and mouse tissues

After the discovery of CA10 in mid 90s the expression of its mRNA was studied in the human brain and in several other tissues (Okamoto et al., 2001). The NB analysis revealed the presence of 2.8 kilo base (kb) band which corresponded to a full length CA10 sequence. The Dot blot (DB) analysis of CA10 mRNA exhibited a low but

24 significant signal in the whole brain and in all parts of the CNS, including the amygdala, cerebellum, cerebral cortex, frontal lobe, hippocampus, medulla oblongata, occipital lobe, putamen, substantia nigra, temporal lobe, thalamus, nucleus accumbens, and spinal cord. The DB technique could not detect any signal for CA10 in the human fetal brain. However, a more detailed analysis indicated the appearance of CARP X protein in the neural cells of the cortex at day 141 of fetal brain development (Taniuchi et al., 2002a). Expression analysis of CA10 was also done using RT-PCR in a panel of human tissues displaying a very strong expression in the brain, kidney and salivary gland and a weak expression in the pancreas, liver, mammary gland and testis (Okamoto et al., 2001). Tashian´s group has reported the presence of many ESTs for CA10 in the human placenta (Tashian et al., 2000). Immunohistochemical staining of CARP X showed a positive signal in both the medulla and cortex of the cerebrum. A very strong signal for CARP X was seen in the myelin sheath and weak signals were also seen in the cerebellar PCs and medullar olivary nuclei (Taniuchi et al., 2002a). The tissue distribution of CARP X in the adult mouse and its developmental expression in the brain showed interesting findings (Taniuchi et al., 2002b). The NB and RT-PCR analysis exhibited a signal only in the brain for the presence of CA10. Expression analysis was done in whole mouse embryos at four different stages (E7, E11, E15 and E17) of development. The signal for CA10 was seen at E11, E15 and E17 during the development by RT-PCR and the NB displayed a significant signal only at E11 during the development. The cellular distribution of CARP X by immunohistochemistry showed moderate expression in neural cells of the cerebrum and midbrain. In the cerebellum, both PCs and dental nuclei exhibited moderate signals for CARP X and weak expression was also seen in the molecular layer, ependymal cells and choroid plexus. The RNA in-situ localization studies revealed that Car10 is mainly expressed in the cone bipolar cells of the mouse retina and weak expression was also seen in the amacrine cells and ganglion cell layer of the retina (Kim et al., 2008).

2.4.2 Expression of CARP X in pathological conditions

The cellular distribution of CARP XI in human brain showed a very strong signal in the linear structures which were assumed to represent the axons or myelin sheaths in the cerebellar medulla and the brain stem (Taniuchi et al., 2002a). These findings prompted the authors to study in more detail whether CARP X is localized in the axon or myelin sheath. The brain sections of a patient with acute disseminated encephalomyelitis were stained with CARP X antibodies and there was no signal for CARP X in the demyelinated axons. Further, CARP X was located to the cytoplasm of neuroblastoma cells (NH12). Similar staining was done in shiverer mouse which had a defective myelin basic protein, and as a result the myelinization was incomplete

(Mikoshiba et al., 1995). The immunohistochemical staining of the shiverer mouse brain showed a complete absence of CARP X in the myelin sheath. The results of the above studies indicate that the CARP X protein is expressed in the myelin sheath. The expression of CARP X in the myelin sheath in the normal human and mouse brain and the loss of expression in the disease suggests the involvement of CARP X in myelin sheath organization (Taniuchi et al., 2002a). The 5’-untranslated region of CA10 gene contains a CCG trinucleotide repeat in normal humans. The presence CCG repeats in CA10 gene and its specific expression in the myelin sheath tends to indicated that the CARP X could be involved in demyelination disorders (Kleiderlein et al., 1998). The microarray expression analysis shows the up-regulation of CA10 mRNA in several cancers suggesting its involvement in the development of neuroblastoma and in several other cancers in humans and other pathological conditions (Kilpinen et al., 2008).

2.4.3 Catalytic activity and inhibition of mutant CARP X hCARP X lacks the two His at positions 94 and 119 which are essential for binding of a zinc atom. Recently, the His were restored making the CARP X active (Nishimori et al., 2013). The mutated CARP X had 92% of the catalytic activity of hCA II. The enzymatic activity of the mutant CARP X protein was effectively inhibited by acetazolamide with the inhibition constant (KI) of 16 nM.

2.5 General properties of CARP XI

The human CA11 gene was discovered accidentally during the construction of a physical map for cone-rod retinal dystrophy. The identified EST D19S799E mapped close to the distal flanking polymorphic marker D19S412 and was reported as CARP-2 (Bellingham et al., 1998). The EST coded for a novel CARP with 328 aa. The catalytic domain of the predicted protein had a substitution of four aa critical for the catalytic activity of the protein (His94Arg, His96Leu, His119Gln, and Thr199Ser). Simultaneously, the existence of the CARP XI sequence was also reported by two other groups from sheep and human (Fujikawa-Adachi et al., 1999; Lovejoy et al., 1998). The first group unexpectedly identified a 1331 bp cDNA while screening a sheep brain cDNA library for frog corticotropin-releasing factor-like peptide sauvagine (Lovejoy et al., 1998). The predicted protein of 328 aa had a theoretical mass of 36 kDa with a signal sequence, suggesting that this protein was secreted. The protein had seven aa residues that are found in sauvagine, but the primary structure of this novel protein was similar to α-CAs and was named as CARP XI. The second group obtained a full length CA 11 cDNA from the human pancreas based on the existence

26 of partial cDNA sequence from dbEST (accession Nos. AA297055 and AA297730) (Fujikawa-Adachi et al., 1999). The cDNA clone was sequenced containing 1475 bp and predicted to encode 328 aa with a mass of 36,200 Da. The deduced aa sequence showed 42-53% similarity to the active CAs and was devoid of catalytically important His residues. Genetic analysis revealed that the CA11 gene was located on chromosome 19q13.2-3. The 3’-UTR had a putative polyA cleavage signal (ATTAAA). NB exhibited a signal for 1.5 kb transcript in the brain, particularly in the cerebellum, cerebral cortex, and putamen.

2.5.1 Expression of CARP XI in normal tissues

An expression pattern of CARP XI was first completed in a panel of human tissues using NB (Bellingham et al., 1998). A strong signal for the CA11 gene product was seen in brain in the region of 1.6 kb in addition to the less intense transcripts of 2.6 and 3.5 kb. The 1.6 kb transcript was the expected size of CA11 cDNA. The analysis also displayed lower intensity bands in the spinal cord and thyroid gland and faint signals were seen in the heart, skeletal muscle, kidney, pancreas, ovary, small intestine, mucosal lining of the colon, peripheral blood leukocyte, stomach, lymph node, trachea and adrenal gland (Bellingham, et al. 1998). In another study, NB analysis showed a presence of 1.5 kb CA11 transcript in the human brain including the cerebellum, cerebral cortex, medulla, spinal cord, occipital pole, frontal lobe, temporal lobe and putamen (Fujikawa-Adachi et al., 1999). No signal for CA11 was detected in heart, placenta, lung, liver, skeletal muscle, kidney and pancreas. However, RT-PCR analysis indicated the expression of CA11 mRNA in the pancreas, liver and kidney, suggesting low levels of expression in these organs. The expression pattern of CARP XI using immunohistochemistry has been studied in the fetal brain at five different gestational periods (Days 84, 95, 121, 141 and 222) and in different parts of the adult brain (Taniuchi et al., 2002a). The presence of CARP XI was seen in neuroprogenitor cells in the subventricular area at day 84 of gestation and in neural cells and choroid plexus at day 95. In the adult brain, a strong signal for CARP XI was seen in the choroid plexus and pia arachnoid. Neurons and astrocytes exhibited a moderate expression of CARP XI. Although the exact function of CARP XI in the brain and especially in the developing brain is uncertain, the expression pattern suggests certain roles in the early development of the brain or differentiation of neuroprogenitor cells. The cellular distribution of CA11 has been analyzed in both adult mouse tissues and embryos of different developmental stages (Taniuchi et al., 2002b). NB analysis revealed the presence of six different bands (1.4, 1.6, 2.4, 3.4, 3.8, and 4.9 kb) in the brain, lung and liver. Among the six bands observed, the 1.5 kb band was the strongest one in the brain. A moderate signal for a 2.4 kb transcript was seen in the heart, skeletal muscle and kidney. The developmental expression of CA11 mRNA was studied in whole mouse embryos at four different gestational periods (E7, E11,

E15, and E17) using RT-PCR and NB. The RT-PCR results suggested the presence of CA11 mRNA at all the stages of development. NB showed the appearance of CA11 mRNA at E7 stage of development and the CA11 signal level decreased as the development progressed. In mouse brain, moderate signals for CARP XI protein were seen in neural cells of the cerebrum, Ammon’s horn and midbrain as well as in the molecular layer, PCs and dental nuclei of the cerebellum, ependymal cells and choroid plexus(Taniuchi et al., 2002b).

2.5.2 Expression of CARP XI in pathological conditions

The expression analysis of CARPs in cancer tissues and in cultured cells has been reported previously (Akisawa et al., 2003; Miyaji et al., 2003). These studies suggested that CARP VIII plays some role in the lung and colorectal cancers. As a continuation to previous studies, the authors studied the expression pattern of all the CARPs in gastrointestinal stromal tumors (GISTs) and in-vitro in cultured GIST-T1 cells (Morimoto et al., 2005). The immunohistochemical analysis of 22 GIST samples presented a very strong signal for CARP XI in 20 (91%) GIST specimens. To understand the role of CARP XI in the GISTs, further studies were performed in GIST-T1 cultured cells transfected with CA11 cDNA (GIST-T1-CA11). The expression level of CA11 mRNA and CARP XI protein showed a remarkable increase in the cells transfected with CA11 compared with the GIST-T1 control cells. Interestingly, a very strong signal for CARP XI protein was observed in the cytoplasm of GIST-T1 cell bodies. There was a significant increase in cell proliferation and cell invasion in GIST-T1-CA11 cells compared with GIST-T1 cells, suggesting that the CARP XI plays a role in the development and spread of GIST tumors. Additionally, the expression pattern of the human CA11 gene at GeneSapiens bioportal indicates the upregulation of CA11 mRNA in several cancers and in other pathological conditions (Kilpinen et al., 2008). Recently, expression of CARP XI was studied in cultured neuronal cells expressing mutant ataxin 3 and in humans and mice with defects in ataxin 3 protein (Hsieh et al., 2013). It is well known that ataxin 3 contains CAG trinucleotide repeats and the mutations causing the expansion of CAG repeats cause a neurodegenerative disorder known as spinocerebellar ataxia 3 /Machado-Joseph disease (SCA3/MJD) in both humans and mice (Kawaguchi et al., 1994). Preliminary studies on the gene expression pattern of human neuroblastoma cells expressing mutant ataxin 3 showed upregulation of CA11 mRNA (Hsieh et al., 2013). The results of the study led to further exploration of expression pattern of CA11 gene in cultured neuronal cells transfected with mutant ataxin 3 gene and in a human patient with SCA 3 and in a transgenic mouse model with MJD. The results of the in-vitro study revealed an altered cellular localization of CARP XI in the cells expressing mutant ataxin 3 compared with the cells expressing WT ataxin 3 (Hsieh et al., 2013).

28 2.5.3 Catalytic activity and inhibition of mutant CARP XI

Mammalian CARP XI lacks all the three histidine residues required for the coordination of the Zn2+ at the active site making it catalytically inactive (Fujikawa- Adachi et al., 1999; Lovejoy et al., 1998; Supuran, 2008). Recently, the catalytic activity of hCARP XI was restored by replacing the three missing histidines. (Nishimori et al., 2013). The mutated protein exhibited a very high catalytic activity. The catalytic activity of mutated protein was 82.6% of the hCAII and was effectively inhibited by acetazolamide, with the inhibition constant (KI) of 29 nM.

3 Aims and objectives of the study

The CARP VIII, X and XI sequences are highly conserved and, in human and mouse are, predominantly expressed in the brain, suggesting important physiological roles in higher organisms. In the present work, we planned to study the distribution of CARP sequences across the species and study the role of CARPs using zebrafish as a model organism as there are no knockout mouse models for CARP genes. In recent years, zebrafish has become an important model organism for studying the embryonic development which is rather similar to the humans. Besides, zebrafish embryos are transparent and develop externally and therefore can be manipulated easily.

The specific aims of the study were to:

Identify and evaluate the distribution of CARP genes in different taxonomic classes (across the species) and study the expression pattern of all the three CARPs in mouse, a mammalian model organism. (I)

Study the conserved residues of CARP VIII on a three dimensional protein structure and visualize the sites of pathogenic mutations in human CARP VIII. (II)

Elucidate the role of CARP VIII in zebrafish during embryonic development, another vertebrate animal model. (III)

Analyze the sequences and conserved residues of CARP X and CARP XI in vertebrate species and study their role in zebrafish embryonic development. (IV)

30 4 Materials and methods

4.1 Bioinformatic analysis of CARP VIII, X and XI sequences (I)

CARP sequences were retrieved from different databases (Ensembl, UniProt, and RefSeq) and at NCBI using Basic Local Alignment Search Tool (BLAST) search (Altschul et al., 1997; Pruitt et al., 2007). Further CARP sequences were generated using mammalian CARPs as query sequences for blast like search (BLAT) from the completed sequence of genomes (Kent, 2002). The obtained sequences were taken through the iterated cycles of multiple sequence alignment (MSA), evaluation and revision (Larkin et al., 2007). For revision of sequences, GeneWise was used for gene model generation with the genomic sequences from the University of California Santa Cruz (UCSC) Genome Browser for the sequences with imperfect matching and for the missing regions in the sequences (Birney et al., 2004; Kent et al., 2002). Gene models generated were confirmed using EST and mRNA sequence data, and to discover and assemble CARPs from less than genome-wide sequenced organisms. In total, we collected 84 full length CARP sequences for further analysis. ClustalW was used for MSA of all the CARPs and the MSAs were visualized using GeneDoc software (Higgins et al., 1996; Nicholas, 1997). The molecular and evolutionary genetic analysis (MEGA) software (version 4.1), was used for the construction of a phylogenetic tree (Tamura et al., 2007). The evolutionary relationship of the CARPs was studied using Neighbor-Joining (NJ) method. A bootstrap test was performed using 1000 replicates and evolutionary distances were computed using the Poisson correction method with the complete deletion option.

4.2 Expression of CARP genes (I, III, and IV)

4.2.1 Extraction of mRNA from mouse and zebrafish

Tissue samples of adult mice were harvested and transferred to the tubes containing RNAlater (Ambion, Austin, TX, USA) (I). Similarly, tissue samples from different organs of adult zebrafish and embryos were collected in RNAlater (III, IV). RNA was isolated using RNeasy kit (Qiagen, Hilden, Germany).

Table 2. Primers for the study of ca8, ca10a and ca10b zebrafish genes (III, IV) Gene Primer Name Upstream (5’-3’) Downstream (5’-3’) primers Size (bp) ca8a ca8-G-DNA GTATTGTGATACATCAGTTT 400 TTAAGCCAAACTGAGACAGG ca8-RT-PCR TTCACTGGGGCAAAGAAAAC 100 ATGGCCTTCAGACCAAGATG ca8-RT-qPCR CCCATGTTAAAGGAGCAGAGCTT - GGTTGGGTCGGTCTAAAGTTGT ITPR1-RT-qPCR CGATAACCCCGGGTGAATG - CCTGGGAGCTTTCAATGTTTG ca10aa ca10a-G-DNA TCAGGGAAGCCACCCTTGAATGA 400 AGAGAAGGCCTGGCCATTGAGG ca10a-RT-PCR ATGGATATAATCTGGGAAATATTTATTATTC Full CTATTTCAGCAGCCATTCGTTC Lengths ca10a-RT-PCR TCTCTCGGGGCTAAACATTG 100 TTGACTTAGCAGTCGCATGG ca10a-RT-qPCR GAAACCAGTCGCATGATTTTTG - TTGCGTTGCCCTGCATT ca10ba ca10b-G-DNA TAGTGTAAACGGGGCCTTACATTTTA 400 GATTGTCCTCACTGCCAAAGTG ca10b-RT-PCR ATGCCGCACGTTTGGGAGTT Full TTATTTCAGAAGCCATTCATTCACTC Lengths ca10b-RT-PCR TGGGGCTGAATATTGAGGAG 100 GGCTGGTTTTGACTGAGGAG ca10b-RT-qPCR CCTGACACCACTGAGGCTCAA - CGGCCGGTGTTGTACATTG CA10b CA10-PCR CGCCTCGAGATGGAAATAGTCTGGGAGGTGCT Full CCGGGATCCCTACTTGAGGAGCCATTCATT Lengths CA11b CA11-PCR CGCTCTAGAATGGGGGCAGCTCGTCTG Full CCGGAATTCTCAGCGACCATGGGGGACACC Lengths aZebrafish sequences, bhuman sequences for rescue of morphant zebrafish

4.2.2 Synthesis of single strand cDNA and RT-qPCR cDNA was synthesized using first Strand cDNA Synthesis kit (High-Capacity cDNA RT Kits, Applied Biosystems, Foster City, CA). The primers were designed using Primer Express® Software (version 2.0) (Applied Biosystems) for the mouse CARPs genes deposited in the Gene Bank (Table 3). Similarly, primers for zebrafish ca8 (ENSDART00000057097), itpr1a (ENSDART00000149019), ca10a (ENSDART00000074540), and ca10b (ENSDART00000055264) were designed using the cDNA sequences from ENSEMBL database (Table 2). RT-qPCR was performed using SYBR Green PCR Master Mix Kit according to the instructions (Applied Biosystems) using the initial denaturation step at 95ºC for 10 min followed by 40 cycles at 95ºC for 15 sec and elongation time of 1 min at 60ºC. Every PCR was performed in a total reaction volume of 15 µl containing 2 µl of the first strand cDNA (20 ng cDNA), 1x Power SYBR Green PCR Master Mix, and 0.5 μM of each

32 primer. The ﬁnal results, expressed as the N-fold relative difference (ratio) in gene expression between the studied samples, internal control gene β-actin and the relative expressions were calculated according to Pfafﬂ’s equation (Pfaffl, 2001). Each experiment was repeated thrice and average of the three values was taken.

Table 3. Mouse primer sequences used for RT-qPCR of mouse CARP genes (I)

Gene Primer Sequence RefSeq numbers Car8 Forward 5’cgggattactgggtctatgaagg3’ NM_007592 Reverse 5’ggctgggtaggtcggaaattgtc3’ Car10 Forward 5’gagagcaagagcccagaactc3’ NM_028296 Reverse 5’ctcaccagtggcagaaatggc3’ Car11 Forward 5’gccggctctgaacaccagatc3’ NM_009800 Reverse 5’gaggaggcgactgaggaatgg3’

4.2.3 Immunohistochemistry of CARPs in mouse and zebrafish (I, III)

The tissue specimens were ﬁxed by immersion in 4% paraformaldehyde (PFA) in phosphate buffered saline (PBS). Samples were dehydrated in an alcohol series, treated with xylene, embedded in paraffin wax and 5 μm sections were cut and placed on Superfrost microscope slides. The paraffin was removed from the sections with xylene and the rehydrated sections were boiled in sodium citrate (0.01 M, pH 6.0) for 20 min and cooled down. The staining protocol was started by incubating the sections in methanol + 3% H2O2 for 5 min, rinsed with 1x Tris-buffered saline (TBS), pH 8.0, containing 0.05% Tween and blocked with Rodent Block M™ (Biocare Medical, Concord, CA) for 30 min. The samples were then incubated with primary rabbit anti-human CARP VIII, CARP X, and CARP XI antibodies ( Catalog # sc-67330, sc-67332 and sc-67333 respectively, Santa Cruz Biotechnology, Inc. Bergheimer Heidelberg, Germany) at a dilution of 1:350 in 1% bovine serum albumin (BSA) for 1 h at room temperature and rinsed with TBS containing 0.05% Tween (The specificity of the antibodies was verified by running a WB using lysate of mouse brain which gave a single band of expected size). The rabbit IgG was used as a control antibody and incubated with 2.5 ml of Rabbit (horseradish peroxidase) HRP-Polymer + 1-2 drops of XM Factor™ (Biocare Medical) for 30 min before rinsing with 1xTBS containing 0.05% Tween and the samples were treated with 3’- diaminobenzidine tetrahydrochloride (DAB) solution for 5 min prior to rinsing with dH2O. Counterstaining was done with Mayer´s Hematoxylin for 1 to 3 sec and rinsed under tap water for 10 min. The slides were mounted with Entellan Neu™ (Merck; Darmstadt, Germany), examined and photographed using a microscope (Nikon Microphot-FXA, Japan). The immunoﬂuorescence staining was performed according to the following protocol: (i) incubation of the samples for 30 min in PBS containing 0.1% BSA, (ii) incubation with rabbit anti-human CARP VIII antibody (Santa Cruz Biotechnology,

Inc., Bergheimer Germany) diluted 1:20 for 1 h or normal rabbit serum diluted 1:20 in 0.1% BSA-PBS, (iii) rinsing of the samples three times for 5 min with 0.1% BSA- PBS, (iv) incubation for 1 h with 1:100 diluted Alexa Fluor 488 goat anti-rabbit IgG antibodies (Molecular Probes, Eugene, OR) in 0.1% BSA-PBS, and (v) rinsing of the samples twice 5 min each with 0.1% BSA-PBS and once with PBS. The stained sections were analyzed and photographed using a Zeiss LSM 700 Confocal laser scanning microscope.

4.2.4 Bioinformatic analysis of CARP VIII (III)

The CARP VIII sequences for the zebrafish (ID: ENSDARP00000057097), mouse (ID: ENSMUSP00000095891) and human (ENSP00000314407) were obtained from the database and ClustalW was used for MSA for calculating the percent identity of each aa. For CA8 gene analysis, the transcripts were retrieved from Ensembl 2012 (zebrafish: ENSDART00000057098), (mouse: ENSMUST00000098290) and (human: ENST00000317995) and the exon structure analysis was done. For co-evolution analysis the ITPR1 sequences used in the analysis were comprised of the region which binds to CARP VIII (Hirota et al., 2003), formed by four residues at the N-terminal and seven residues at the C-terminal region (corresponding to aa 1368–1649 in human ITPR1 isoform 1). Similarly, the CARP VIII sequences had the minimal ITPR1-binding fragment 45–291 (human numbering) and the sequences were chosen from the species which were identical for both proteins. The CARP VIII alignment had 31 sequences, while the alignment of ITPR1 included 37 due to an early duplication in the ray-finned fish lineage which has led to two co-orthologs of ITPR1 (itpr1 a and itpr1 b) genes in fish genomes. A phylogenetic tree was reconstructed using the CARP VIII alignment with NJ method using fish sequence as the outgroup. Two alignments were built from the ITPR1 sequences so that the 25 tetrapod species were present in both, whereas the 12 sequences from 6 fish species were divided. The orthologs of zebrafish itpr1a were included in one alignment (‘clade 1’) and the orthologs of itpr1b in another alignment (‘clade 2’). The two alignments of ITPR1 from the 31 species were analyzed separately for coevolution with the CARP VIII sequences. The CAPS program (Fares and McNally, 2006) (PMID: 17 005 535) was used with the MSAs and the phylogenetic tree to identify the likelihood of coevolution between sites of CARP VIII and ITPR1 proteins.

4.2.5 Bioinformatic analysis of CARP X and XI sequences (IV)

For sequence and phylogenetic analysis, 83 CARP X and 54 CARP XI sequences, and their corresponding coding regions of transcript sequences were obtained from

34 Ensembl. Due to the unique position of the jawless vertebrates in evolution, the two L. japonicum CARP X-like sequences which lacked an initiation codon (ATG) were also included in the analyses. The two sequences from D. melanogaster CARPs were RefSeq NM_132179 (“CARP-A”) and NM_001258664 (“CARP-B”) and a cah-2 of C. elegans (RefSeq NM_063166.6) were used for comparison. The sequences were aligned using Clustal Omega (Sievers et al., 2011). To calculate amino acid identity percentages, the number of conserved amino acid residues in each aligned sequence pair was divided by the length of the shorter sequence. SignalP 4.1 (Petersen et al., 2011) and TargetP 1.1. (Emanuelsson et al., 2000) were used to predict signal peptides in the proteins. The default cutoff values were used in SignalP (chosen to optimize the performance), and predefined cutoffs for specificity >0.95 were used in TargetP. The C-terminal glycosylphoshpatidylinositol (GPI) anchor sites were predicted in the Pred-GPI server (Pierleoni et al., 2008) with the general model and taking “Highly probable” and “Probable” predictions (99.5% specificity cutoff) as positive. Potential N-glycosylation sites were identified by the sequence motif N – not P – S/T.

For the phylogenetic tree the 57 vertebrate CARP X/XI protein sequences were aligned with Clustal Omega and transformed into a codon-based cDNA alignment with the Pal2Nal web server (Suyama et al., 2006) with the remove gaps/internal stop codons option. MrBayes (Ronquist et al., 2012) was run to estimate a reliable set of model parameters. The tree was obtained using GTR+I (+G) model after 50,000 cycles. A 50% majority rule consensus tree was created, rooted by Archaeopteryx (Han and Zmasek, 2009) with the Drosophila sequence CARP-B (CG32698) as an outgroup. Final trees were drawn aided by the R package Ape (Paradis et al., 2004).

4.3 Molecular analysis of CARPs in zebrafish (III, IV)

4.3.1 Zebraﬁsh husbandry for the knockdown experiments (III, IV)

The WT zebraﬁsh of the AB strain were maintained at the zebrafish facility of the IBT. Embryos were collected from the breeder tanks and rinsed with embryonic medium (5 mM NaCl, 0.17 mM KCl, 0.33 mM CaCl2, 0.33 mM MgSO4 and 10–15% Methylene Blue) (Sarsted, Nümbrecht, Germany), transferred to Petri dishes and staged according to standard protocols (Kimmel et al., 1995). The embryos were kept in Petri dishes (50 embryos per dish) in embryonic medium at 28.5ºC. We used 0-5 dpf embryos for the knockdown of CARP genes using antisense MOs and hence, they did not require any ethical permission from the ethical committee. (http://www.laaninhallitus.fi/lh/etela/hankkeet/ellapro/home.nsf/pages/indexfin).

4.3.2 Sequencing of zebrafish CARP genes (III, IV)

Sequencing of ca8, ca10a, and ca10b genomic regions was performed to analyze polymorphism in the genomes. Similarly, sequencing of cDNAs of ca8, ca10a, and ca10b from zebrafish was also done using the primers presented in table 2. In a 10 µl reaction, we took 2 µl BigDye (BigDye® from Life Technologies), 2 µl sequencing buffer, 0.5 µl of 3.5 µM forward or reverse primer, 1 µl template DNA and 4.5 µl of dH2O. The parameters used for PCR were: denaturation 96ºC for 10 sec, annealing at 50ºC for 5 sec and extension at 60ºC for 4 min with 25 cycles. PCR products were transferred to 1.5 ml tube containing 32 µl of dH2O and 8 µl of 95% ethanol and the DNA was precipitated by keeping the tubes at -20ºC for 30 min. The DNA was pelleted by centrifugation at 16,000 x g and washed with 70% ethanol and the DNA was sequenced.

4.3.3 Knockdown of CARP genes using morpholinos (III, IV)

Each zebrafish CARP gene was knocked down using two different antisense MOs. The antisense MOs and MOs for Tumor protein p53 (p53) gene and Random Control (RC) MOs were obtained from GeneTools (Table 4) (GeneTools LLC, Philomath, OR). ca8 gene was knocked down by MO1 which blocked the starting codon, ATG. The MO2 targeted the ca8 gene at a splice site junction resulting in knocking down of one of the exons of ca8 mRNA. For ca10a gene, we used a translational blocking MO (MO1) and a splice site blocking MO (MO2), but for ca10b gene both the MOs targeted two different exons. The RC-MOs were used as control MOs and p53 MOs were used to suppress the p53 mRNA. The MOs were resuspended in dH2O at a 1 mM stock concentration. For the injections, the MOs were diluted to the intended concentration with 0.2 M, KCl, 1% phenol red (Sigma, Poole, UK) and 10% rhodamine B (Sigma-Aldrich) dyes were added to the diluted MOs to monitor the injection of morpholinos. We injected 1 nl of MOs into the yolk of one- to two-cell stage larvae. When p53 MO was included in the injections, the concentration of p53 MOs was one and a half times of the concentration of antisense MOs for ca8, ca10a or ca10b.

4.3.4 Analysis of CARP VIII protein and CARP mRNAs (III, IV)

For Western blotting (WB), morphant and WT zebrafish larvae were homogenized with a hand held pestle in a microcentrifuge tube with 50 µl of lysis buffer (50 mM Tris–HCl pH 7.4, 150 mM NaCl, 5 mM ethylene-diamine-tetra-acetic acid (EDTA), 1% NP40). The lysates were centrifuged at 10 000 x g for 5 min, 10 µl of the lysate was used per lane on a 10% sodium dodecyl sulfate (SDS) polyacrylamide gel along

36 with a protein marker (Bio-Rad, Hercules, CA) and ran the gel at 100 V for 10 min and then 200 V for 40 min. Separated proteins were transferred onto a polyvinylidene diﬂuoride (PVDF) membrane (Immobilon, Millipore). CAPR VIII protein was detected by incubating the membrane with primary rabbit anti-human CARP VIII antibodies (1:50) (Santa Cruz Biotechnology) and horse radish peroxidase-conjugated goat anti-rabbit IgG (H + L) antibody. The signals were visualized using a chemiluminescence system (Western Lightning; Perkin Elmer Life Science). The total RNAs were isolated from 5 dpf WT and morphant fish, cDNA was prepared and PCR amplification was done as described above. The amplified products were separated on agarose gel and the bands were visualized and photographed.

Table 4. Morpholino sequences used to knock CARP genes down (III, IV) Gene Name of morpholino Sequence of morpholino ca8 MO1-Translation blocking 5′CGTGGAGAGAAGATTGAGTTGCCAT3′ MO2-Splice site blocking 5′AGGATCATGCTGAAATCCAAATCTG3′ ca10a MO1-Translation blocking 5’TGTCTTCATTCCAAGTCCATTGCGC3’ MO2-Splice site blocking 5’CATCTGTAAGGACAAGCAGAGGTTT3’ ca10b MO1-Splice site blocking 5’ACTGACCTGAAAAACACACCCAAAC3’ MO2-Splice site blocking 5’GACTGCATCTATGGAAATTCATTAT3’ RC Control MO 5’CCTCTTACCTCAGTTACAATTTATA3’ p53 p53 MO 5’GCGCCATTGCTTTGCAAGAATTG3’

4.3.5 Live image analysis of zebraﬁsh phenotypes (III, IV)

For phenotypic analysis the larvae were anesthetized using 5% Tricaine in embryo medium and embedded in 17% high molecular weight methyl cellulose on a transparent Petri dish. The behavioral studies of morphant larvae injected with antisense MOs were studied by analyzing the swim pattern of 5 dpf morphant larvae and comparing the swim pattern with the WT larvae. Morphant larvae were transferred (5 per well) into a 15×30 mm one-well Petri dish containing embryo medium and placed on a light stage with a transmitting light source. The movement of the larvae was observed for 10 min while simultaneously tracing the movements. Similarly, a 30s video of swim pattern was recorded for each group of larvae and images of 0-5 dpf larvae were taken using a Lumar (version1.12) ﬂuorescence stereomicroscope (Carl Zeiss MicroImaging GmbH) and AxioVision software (versions 4.7 and 4.8). Each experiment was repeated a minimum of three times.

4.3.6 Histochemical analysis of morphant and control larvae (III, IV)

For histochemical analysis, 5 dpf morphant and WT zebraﬁsh larvae were washed with PBS and ﬁxed with 4% PFA in PBS for 3 h at room temperature. The larvae

37 were then transferred to 70% ethanol and stored at 4ºC. The larvae were placed on a paraffin block containing 1% low melting agarose and oriented dorsal side up before embedding in parafﬁn. Fixed sections of the larvae were deparafﬁnized in xylene, rehydrated in an alcohol series and histological staining was done with Mayer’s hematoxylin and Eosin Y (Sigma-Aldrich). After dehydration, sections of the larvae were mounted with Entellan NeuTM (Merck, Darmstadt, Germany). Stained slides were examined and photographed using a Nikon Microphot microscope (Nikon Microphot-FXA, Japan).

4.3.7 Assay to detect apoptotic cell death in morphant fish (III, IV)

The morphant and WT zebrafish larvae were fixed in 4% PFA in PBS and stored in 70% ethanol. Fixed samples were then embedded in paraffin and sections of 5 µm were cut by a microtome. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay was performed using a QIA39 FragEL TMDNA Fragmentation Detection Kit, Fluorescent-TdT Enzyme (Merck Chemicals Ltd., Nottingham, UK). Proteinase K-treated and deparafﬁnized sections of larvae were incubated with TdT enzyme followed by incubation with anti-digoxigenin. Positive signals were detected by ﬂuorescence microscopy and photographed using a Nikon Microphot microscope (Nikon Microphot-FXA, Japan). The assay was repeated twice for each zebrafish sample.

4.3.8 Electron microscope analysis (III)

Electron microscopy was used to study the ultrastructural changes in the cerebellum of ca8 morphant fish. For electron microscopic study, the 5 dpf morphant zebrafish injected with 125 µM ca8-MO1 were ﬁxed with 2% glutaraldehyde, 2% formaldehyde in 0.1 M sodium cacodylate buffer, pH 7.4 at 4ºC overnight. The rest of the procedure was carried out at the Electron Microscopy Unit, Institute of Biotechnology, University of Helsinki in collaboration with Dr. Eija Jokitalo. The samples were post-ﬁxed with 1% reduced, buffered osmium tetroxide for 1 h at room temperature. The samples were dehydrated with a graded ethanol series and embedded into TAAB embedding resin (TAAB Ltd., UK). Semi-thin sections of the samples were cut and stained with Toluidine blue for light microscopy analysis. A suitable area was then selected for ultra-thin sectioning, and the sections were collected on pioloform-coated single-slot copper grids and post stained with uranyl acetate and lead citrate. The resulting sections were analyzed using a transmission electron microscope (Tecnai 12, FEI, Hilsboro, OR) operated at 80 kV, and images were acquired with a MultiScan 794 1K × 1K CCD camera (Gatan, Pleasanton, CA). In all comparisons, at least six larvae for each category were examined.

38 4.3.9 Cloning of human CA10 and CA11 genes in pcDNA3.1

Human CA10 and CA11 obtained from IMAGE (MGC Geneservice Ltd, Cambridge, UK) and were inserted into plasmid pcDNA3.1. The plasmid containing the inserts was cloned in One Shot® TOP10 E. coli cells (Invitrogen) by taking 1µl of the plasmid+25 µl competent cells and incubated on ice for 30 min. The cells were heat shocked at 42ºC for 30 sec and transferred to the ice for 2 min 125 µl of SOC medium was added to each tube and kept at 37ºC in a shaker at 225 rpm for 1 h. 20 µl of the cells were spread on (Luria-Bertani) LB agar plates and incubated at 37ºC for 16 h. The colonies were screened for the presence of inserts using the PCR conditions: denaturation at 98ºC for 2 min, 35 cycles of denaturation at 98ºC for 10 sec, annealing at 55ºC for 30 sec, extension at 72ºC for 1 min and extension at 72ºC for 10 min. Plasmid DNA was isolated from 3 ml of overnight culture using QIAprep Spin Miniprep Kit (Qiagen. Hilden, Germany) For synthesis of capped mRNA, the plasmid was digested with KpnI and the linearized plasmid was treated with proteinase K (100-200 µg/ml) and 0.5% SDS for 30 min at 50ºC dry bath. To extract the DNA template, a mixture of phenol, chloroform and isoamyl alcohol (25:24:1) was added and vortexed for 20 sec and centrifuged at 16,000 x g for 5 min. The upper aqueous phase was carefully transferred to a fresh tube and the DNA was precipitated with 200 µl 95% ethanol at -80ºC for 1 h. The tubes were centrifuged at 4ºC for 30 min at 16,000 x g to pellet the DNA and was washed with 150 µl of 70% ethanol and centrifuged for 2 min at 4ºC at 16,000 x g. The DNA obtained as above was resuspended in 30 µl sterilized dH2O. The DNA was quantitated using NanoDrop spectrophotometer.

4.3.10 Synthesis of mRNA and rescue of ca10a and ca10b morphants

The transcription of CA10 and CA11 genes was performed using mMESSAGE mMACHINE® T7 Kit from Ambion (Life technologies). A 20 µl reaction mixture (10 µl 2XNTP/CAP, 2 µl of 10X reaction buffer, 1 µg of the linear DNA template, 2 µl of enzyme mix and 5 µl of nuclease free water) was incubated at 37ºC for 1 h. After the incubation, 1 µl of TURBO DNase was added to the tube, mixed well and incubated at 37ºC for an additional 15 min. The capped RNA was recovered from the reaction mixture by adding 30 µl of Lithium Chloride (LiCl) to precipitate the RNA. The reaction mixture was chilled at -20ºC for 30 min and centrifuged at 4ºC for 15 min at 16,000 x g to pellet the RNA. The RNA pellet was washed with 1 ml of 70% ethanol and the RNA was resuspended in nuclease free dH2O and quantitated using NanoDrop spectrophotometer. For the rescue work, the embryos were divided into two groups. The first group of embryos was injected with 300 µM splice site blocking MOs and the second group co-injected with 80 pg of capped mRNA and 300 µM antisense MOs per embryo.

5 Results

5.1 Distribution and expression of CARP VIII, X and XI sequences

5.1.1 Distribution and evolutionary analysis of CARP sequences (I)

The number of CARPs has been increasing steadily with the completion of genome sequencing projects. Our bioinformatic analysis suggested that the annotation of CARPs is incomplete. The purpose of this study was to identify all the CARP sequences, evaluate their distribution across the species, and study their evolutionary relationship. We identified 84 CARP sequences from 38 organisms including 35 vertebrates and 3 invertebrates. The CARP VIII group was comprised of 33 sequences which included 3 sequences from deuterostome invertebrates. There were 31 sequences of CARP X and one of them belonged to a chordate (B. floridae). The 19 CARP XI sequences were found only in vertebrates (Table 1, I). The sequence analysis of CARPs (Figs 1, 2, and 3, in I) displayed a minimum of 67% identity among the vertebrate CARP VIII sequences, while the sequence identities among the vertebrate CARP X and XI sequences were at least 70%. The invertebrate sequence identities of CARP VIII and CARP X were 40% to 45% compared with vertebrate orthologs. The evolutionary relationship of CARPs was studied from the phylogenetic tree (Additional file 1, I) and the sequences in the tree showed two groups, group one included CARP VIII sequences, while the other group comprised of CARP X and XI sequences (Fig 4, I). Interestingly, the CARP X sequences formed three subgroups, group one comprised of the sequences from mammals, frog and lizard and the other two subgroups consisted of sequences from fish species. CARP XI sequences were derived from mammals, while CARP XI sequences from lizard and frog formed an out-group. The study suggested an independent duplication of CA10 in the ray-finned fish lineage (actinopterygii), whereas CA11 might have appeared from the second gene duplication after the separation of the actinopterygii and sarcopterygii lineages, or, alternatively, the duplication to CA10 and CA11 might have happened in early vertebrates, with a subsequent loss of CA11 in the ray-finned fish lineage.

40 5.1.2 Expression analysis of CARPs in mouse tissues (I)

The studies on the tissue distribution of CARPs have been rare and produced slightly conflicting results. We studied the systematic distribution of CARPs at both mRNA and protein levels in mouse tissues. The results of RT-qPCR revealed that the Car8 mRNA was predominantly expressed in the cerebellum and a high level of expression was also seen in the liver and lung. Low signal was also seen in the stomach, duodenum, ileum, jejunum, colon, spleen, kidney, heart, frontal cortex, parietal cortex, midbrain, and eye, and a faint signal was seen in the ovary, skeletal muscle, and the testis. Car10 mRNA indicated strong signals in the cerebellum, frontal cortex, and parietal cortex, while midbrain and eye showed weaker expression. The expression of Car11 mRNA was high in the frontal cortex, parietal cortex and cerebellum, and moderate expression was seen in the midbrain. A low signal for Car11 was also seen in the colon, kidney, ovary, heart, and lung. A very faint signal for Car11 was seen in liver, stomach, duodenum, ileum, jejunum, spleen, and eye. The expression of CARP mRNAs is summarized in table 5.

Table 5. Summary of the expression of CARP mRNAs in mouse tissues (I) Tissues Car8 Car10 Car11 Cerebellum +++ +++ ++ Frontal cortex + ++ +++ Parietal cortex + ++ +++ Midbrain + + + Eye + +w +w Liver ++ - - Stomach +w - - Duodenum + - - Ileum + - - Jejunum + - - Colon + - + Pancreas - - - Spleen +w - - Kidney + - + Ovary +w - + Heart + - + Lung ++ - +w Skeletal Muscle - - - Testis - - - Uterus - - - Expression level: +++ Strong, ++ Moderate, + Low, +w Very low, - No signal

The immunohistochemical analysis of CARP VIII displayed a broad expression pattern compared with the expression of CARP X and CARP XI (Figs 8, 9, 10 and 11, I). CARP VIII was predominantly expressed in the cerebellar PCs and cerebrum. Lower levels of expression were also seen in the liver, pancreatic Langerhans islets, submandibular gland, stomach, colon, kidney, and lung. An intense signal for CARP X was seen only in the respiratory epithelium of the lung and a faint signal for CARP

X was seen in the cerebellar capillaries and stomach. A very faint signal was detected in the heart muscle. CARP XI showed positive signals in cerebellum, cerebrum, liver, stomach, small intestine, colon, kidney, and testis. The cerebellar PCs also exhibited a prominent signal for CARP XI.

5.1.3 Comparative analysis of CARP VIII sequence (II, III)

The inactivity of CARPs has led scientists to suggest that CARPs might be involved in unknown functions by interacting with other proteins. Indeed, CARP VIII interacts with ITPR1 and is also associated with motor co-ordination function in mouse and human (Hirota et al., 2003; Jiao et al., 2005; Kaya et al., 2011; Turkmen et al., 2009). Based on the above information we performed further studies on CARP VIII to get better insights into its structure and function. The comparative analysis of hCARP VIII with hCA XIII and hCA II revealed that the CARP VIII sequence contains an additional Glu stretch at the N-terminal end up to ‘WGYEE’ encoded by the first exon (Fig 2, II). In CARP VIII, most active site residues are conserved except for His 94 and Gln 92, which are substituted by Arg117 and Glu115, respectively. Comparison of CARP VIII sequences from S. purpuratus showed the first exon is longer than the hCARP VIII sequence by an insertion of 18 residues, and it also suggested the absence of a Glu rich region. The Glu stretch was also found in fish and birds, but the number of Glu residues was fewer than in human sequences. The MSA of CARP VIII was done using sequences from vertebrate and invertebrate organisms, and the sequence identities were calculated from pairwise alignment (Fig 3 and Table 3, II). Results of the analysis indicated higher conservation of sequences among CARP VIII than among the active CAs. Sequence conservation studies (11 CARP VIII sequences were used) of the CARP VIII structure, in comparison with hCA II, showed that a majority of the highly conserved residues in CA II are in the active site and in other structurally important sites of the protein core, while the protein surface exhibited high variability (Fig 6, II). Conversely, CARP VIII displayed a very high level of conservation on the surface. The structural basis of a misfolding of the CARP VIII protein with a mutation of S100P, in comparison with normal CARP VIII, showed the location of Ser 100 at the end of 훽 strand 6 (Fig 11, II). Replacement of Ser with Pro would cause a rigid bend in the peptide backbone that likely prevents β6 and/or the β6/β7 loop from assuming the conformations they have in the WT protein. The shorter and more conserved loop between strands β5 and β6 may amplify the problem in folding of the S100P mutant protein even further (I).

42 5.1.4 Bioinformatic analysis of zebrafish CARPs and their genes (III, IV)

Many human genes have orthologs in zebrafish with similar functions. We found orthologs of CARP genes in zebrafish (I). Therefore, we used zebrafish as a vertebrate model to get insights into the roles of CARP genes. To study the function of the CARP genes, we first used bioinformatic tools to analyze these genes and their corresponding protein products from zebrafish. For analysis of CARP VIII gene (III), we retrieved zebrafish, mouse and human CA8 transcripts and their proteins from Ensembl. The ca8 transcript was encoded by a 23.49 kb gene which is located on the forward strand of the chromosome 2 and had 9 exons, encoding 281 aa. The lengths of the mouse and human CARP VIII transcripts were above 90 kb and were located on reverse strands of chromosome 4 and 8, respectively. In zebrafish, a major portion of exon 1, part of exon 8 and entirety of exon 9 did not code for any aa, similar to the mouse and human genes (Fig 1A, III). The comparative analysis of zebrafish CARP VIII sequences with human and mouse CARP VIII showed a 79% overall aa identity, and the aa identity within ITPR1 binding region (aa 40-290 in human) was 85% (Fig 2A, III). The zebrafish CARP VIII sequence comprised 281 aa compared with mouse (291) and human (290) proteins due to absence of Glu residues at N-terminal end of zebrafish sequence. Sequencing of a zebrafish ca8 gene revealed nucleotide changes at three places without any change in the protein (Fig S1, III). CARP VIII binds to ITPR1 and takes part in the modulation of a Ca2+ channel (Hirota et al., 2003). In the present work (III), we studied the functional association between ITPR1 and CARP VIII throughout the vertebrate evolution. The coevolution analysis of 31 ITPR1 sequences, (6 fish orthologs, version itpr1a and 25 tetrapod sequences) containing the ITPR1 region of 271 aa which interacts with CARP VIII, showed that the two proteins had several significant coevolving sites (P value of <0.001). Similar to the analysis of CARP VIII genes, we have done the analyses of CARP X and CARP XI genes. In zebrafish, the transcript for ca10a is encoded by a 252.67 kb gene located on the forward strand of chromosome 12 and the transcript for ca10b is encoded by short gene (61.86 kb) located on the forward strand of chromosome 3. The zebrafish ca10a and ca10b genes contain 9 exons similar to the human CA10 gene and code for 328 and 326 aa, respectively.

5.1.5 Features of secretory proteins in CARPX-like sequences

Gene prediction analysis of CARP X and XI sequences improved 881 aa positions in incomplete sequences. Similarly, the analysis also restored 10 incomplete sequences to completeness (Table 3 and Supplementary file S1, IV). Sequencing of AB zebrafish strain ca10a and ca10b sequences did not indicate any change at the nucleotide level (data not presented). Zebrafish ca10a and ca10b are orthologs of CA10 gene (I), CARP Xa showing 90% similarity with hCARP X and CARP Xb

43 diverged slightly and has 75% identity with hCARP X (Table 4, IV). In addition to sequence percent identity, phylogenetic tree and MSA support that fish CARP Xa and Xb are closer to tetrapod CARP X than to CARP XI (Figs 1 and 2, IV). The transcript for ca10a is encoded by a 252.67 kb gene located on the forward strand of chromosome 12 and the transcript for ca10b is encoded by short gene (61.86 kb) located on the forward strand of chromosome 3. The zebrafish ca10a and ca10b genes contain 9 exons similar to the human CA10 gene and code for 328 and 326 aa, respectively.

Analysis of CARP X-like sequences revealed the presence of signal peptide in 96% of sequences including the sequences from D. melanogaster and C.elegans. Interestingly, the signal peptide cleavage sites in hCARP X and XI, and zebrafish CARP Xa and Xb occur at the end of the sequences coded by the first exon in each gene (Fig 4, IV). All the protein sequences analyzed in the study showed conservation of three Cys residues at positions 60, 244, and 310 in hCARP X. An additional fourth Cys residue at position 296 hCARP X was conserved in all of the CARP X sequences in tetrapods, coelacanth, lamprey, and in all CARP Xa and CARP Xb, but not in CARP XI (Fig 4, IV). Similarly, two conserved N-glycosylation motifs were seen in CARP X-like proteins at N116 and N168 (hCARP X numbering). These positions corresponded to non-conserved surface residues, which are not part of glycosylation motif (n-not p-S/T) in any other human or zebrafish CAs, except for CARP X/XI/Xa/Xb. The third glycosylation site found in CARP XI was unique and another glycosylation site unique to CARP X/Xa/Xb (Fig 4, IV)

5.1.6 Expression analysis of CARP genes in zebrafish (III, IV)

We studied the expression pattern of zebrafish CARP genes in 0-5 dpf embryos and in a panel of adult zebrafish tissues to get insights into their distribution (Fig 3A, III). The expression analysis of ca8 mRNA showed expression in all tissues analyzed (Table 6). The ca8 gene was predominantly expressed in the brain and moderate levels of ca8 mRNA were seen in the kidney, eye, gills, heart and tail. Low level of expression was seen in the testis, ovary, bowel, liver and gills. At the protein level immunohistochemistry exhibited the presence of CARP VIII in the cerebellar PCs (Fig 3C, III). The expression of ca8 gene during development showed a significant signal at 0 hours post fertilization (hpf) suggesting a maternal origin. The high level of ca8 mRNA throughout development further suggests its requirement during this phase (Supplementary Fig S3A, III). Similarly, we studied the expression of itpr1a mRNA to evaluate if the expression pattern of these genes (ca8 and itpr1a) is the same. Indeed, the expression pattern suggested a significant presence of itpr1a mRNA at 0 hpf similar to ca8 mRNA (Fig S3B, III). Expression analysis of ca10a and ca10b in tissues revealed an intense signal for ca10a mRNA in brain, eye and heart. Low signal for ca10a was also seen in testis,

44 kidney, ovary, skin, spleen, swim bladder, fins, gills, intestine and muscle. No signal for ca10a was seen in the liver (Table 6). The ca10b gene was highly expressed in the ovary and high levels of ca10b were also seen in the brain. Positive signals for ca10b were also found in the swim bladder, spleen, testis and eye. Low level of ca10b was observed in the kidney, intestine, skin, heart, gills and fin (Table 6). The relative expression of ca10a and ca10b genes was studied during embryonic development (Fig 6A and B, IV). High levels of ca10a were seen at 96 hpf followed by 120, 144, and 168. Low level expression for ca10a was seen at 72, 48, 24, 12, and 8 hpf and a very faint signal was also seen at 0 hpf and 4 hpf. These results indicate the requirement of ca10a expression throughout the development and especially after 72 hpf. The presence of ca10b at 0 hpf suggested that the ca10b is of maternal origin. An intense signal for ca10b was also seen at 72, 96, 120, 144 and 168 hpf and faint signal was observed at 12, 24 and 48 hpf (Fig 6B in IV). The pattern of ca10b expression suggests its requirement in early embryonic development.

Table 6. Summary of the expression of CARP genes in zebrafish tissues (III, IV).

Tissues ca8 ca10a ca10b Brain +++ +++ +++ Eye ++ +++ ++ Heart ++ +++ + Muscle +w +w - Swim bladder NA + +++ Skin + + + Spleen NA + ++ Ovary + + +++ Intestine + +w + Gills ++ +w +w Liver + - - Kidney ++ + + Testis NA + ++ Fins ++ + - Expression level: +++ Strong, ++ Moderate, + Low, +w Very low, - No signal, NA-Not analyzed

5.1.7 ca8 knockdown leads to developmental defects in zebrafish (III)

We used two morpholinos, MO1 and MO2, to target translation of mRNA and splice site blocking (exon 4) of ca8 gene, respectively. The RC-MOs were used as controls and p53 MOs were used for correcting the potential off-target effects of antisense MOs. After the injection of MO1 and MO2, we studied the specificity of the MOs first by analyzing the CARP VIII protein (in case of MO1 injections) in the morphant and WT larvae by WB method. The results showed a complete absence of CARP VIII protein from 3 dpf larvae and appearance of a faint band in 5 dpf embryos which corresponded to CARP VIII protein when compared with the WB of lysates from WT embryos (Fig 4, B and C, III). Second, the analysis of ca8 mRNA

45 from morphant embryos injected with MO2 showed a truncated mRNA in addition to a normal length mRNA (Fig D, III), suggesting a partial knockdown of ca8 gene. The CA8 morphants injected with 125 µM of MO1 exhibited abnormal changes in the head as early as 9 hpf (data not presented). The 5 dpf morphant embryos displayed a variety of defects, which included an abnormal head shape, fragile body and curved body axis, small eye size, absence of the swim bladder and otolith sacs, and pericardial edema (Fig 5A, III). The ca8 morphants injected with MO2 demonstrated less severe phenotype (Fig 4 B, III). The ca8 morphant embryos injected with different concentrations of MO1 (ranging from 2 µM to 500 µM) showed a wide range of phenotypic changes from no obvious changes to severe phenotypic defects with increased mortality (Supplementary material, Fig S2 A-G and Table 2, III). The development of RC injected embryos and WT embryos was normal without any phenotypic defects. Similarly, co-injection of p53 MO did not display any improvement in phenotype in 125 µM MO injected morphants.

5.1.8 Suppression of ca10a and ca10b genes leads to phenotypic defects (IV)

There are only a few reports available on ca10a and ca10b expression, these suggest that the genes are primarily expressed in CNS of mouse and human (Nishimori et al., 2003; Taniuchi et al., 2002a; Taniuchi et al., 2002b). In our study (I), the bioinformatics analyses of CARPs showed that the CA10 and CA11 genes are conserved across species and orthologs for these genes are found in zebrafish. Knockdown studies on ca8 (III) encouraged us further to silence the ca10a and ca10b genes in order to get insights into their role in zebrafish. We used two antisense MOs for each ca10a and ca10b gene (Fig 7 A, B and C, IV). The MO1 for ca10a targeted the translation of ca10a mRNA and the MO2 targeted the splice site junction (exon 8), resulting in the expression of a shorter length mRNA (Fig 7 D of IV). The antisense MOs for ca10b gene (MO1 and MO2) targeted splice site junctions, resulting in the truncated ca10b mRNAs (Fig 7 E, IV). The presence of truncated mRNAs for ca10a and ca10b genes suggested that the MOs used were functional for targeting the ca10a and ca10b genes. ca10a morphants injected with 200 µM of MO1 presented less severe phenotypic defects compared with the ca10b morphants injected with a similar concentration of MO1(Fig 8B, IV). The ca10a morphants exhibited defects in the head with abnormal body, small eye size and delayed hatching from the chorion after 1 dpf (Fig 8B, IV). As the development progressed, the larvae showed a long, tapering and curved tail, curved body, pericardial edema, and absence of swim bladder and otolith vesicles. Interestingly, the ca10a morphants injected with MO2 exhibited less severe abnormalities compared with the abnormalities produced by MO1. The ca10b morphants injected with 200 µM concentration of MO1 exhibited an abnormal phenotype at 12 hpf (data not shown). After 1 dpf, the ca10b morphants showed a reduction in the length of the body and larvae were fragile with a high rate

46 of mortality (Fig 8C and Fig 9 and Table 3, IV). The ca10b morphants obtained with the injection of MO2 showed a milder phenotype compared with the phenotype obtained by MO1 injection and displayed abnormal body structure, sizes of the head and eye were smaller, mild pericardial edema, absence of otolith sacs, unutilized yolk sac and curved tail (Fig 8E, IV). The larvae injected with p53 MOs did not show any difference either in phenotype or in the mortality rate (Fig 9 and Table 3, IV). The ca10a and ca10b morphants injected with 300 µM ca10a and ca10b MOs could be partially rescued by hCA10 and hCA11 mRNAs (Fig 11, IV).

5.1.9 Morphological changes and apoptosis in morphant fish (III, IV).

Histochemical staining revealed gross morphological changes in the cerebellum of ca8 morphants (Fig 6 G and H, III) and the TUNEL assay suggested apoptotic cells in the brain (Fig 6 A, B, C and D, in III). Studies on ultrastructural changes using transmission electron microscopy showed increased neuronal cell death. The apoptotic changes included a condensed nucleus, fragmented mitochondrial proﬁles and debris of dead cells (Fig 6 E and F of III). The histochemical studies on ca10a and ca10b morphants displayed gross morphological changes in the head and eye region (data not shown) compared with WT zebrafish larvae. The TUNEL assay on ca10a morphants indicated cells undergoing apoptosis in the head and eye regions (Fig 10 A and B, IV). Massive apoptosis was observed in the head and tail region of zebrafish larvae injected with ca10b-MOs (Fig 10 C and D, IV).

5.1.10 Abnormal swim pattern in ca8, ca10a and ca10b morphants (III, IV)

Earlier reports suggested that a mutation in the CA8 gene is associated with gait disorder in waddle mice and ataxia in humans (Jiao et al., 2005; Kaya et al., 2011; Turkmen et al., 2009). Therefore, we studied whether the knockdown of CARP genes lead to any change in the swim pattern in the morphant embryos. For the swim pattern study, we injected the lowest concentration of antisense MOs at which no apparent phenotypic changes were seen in the morphants. In the ca8 knockdown study (III), we used 5 dpf morphant embryos injected with 2 µM and 6 µM concentrations of ca8-MO1 which did not indicate any apparent physical deformity. Interestingly, the ca8 morphants displayed difficulties in balancing the body with an increased turning angle compared with WT larvae (Fig 7B and movies S1-S4, supplementary material, III). The swim pattern study was also carried out on ca10a and ca10b morphants injected with 50 µM MOs, which did not suggest any apparent physical deformities. The 5 dpf morphant larvae injected with ca10a and ca10b MOs exhibited clear change in the swim pattern compared with RC control larvae and WT larvae (Supplementary data, Movies 1-3, in Fig 12 and Fig 13 A-D of IV).

6 Discussion

6.1 Bioinformatic and molecular analysis of CARP sequences

6.1.1 The CARPs are widely distributed across species (I, IV)

Previous studies on the occurrence of CARP sequences were limited to few species (Bellingham et al., 1998; Fujikawa-Adachi et al., 1999; Kato, 1990b; Okamoto et al., 2001). Therefore, one aim of this study was to analyze the distribution of CARP sequences in multiple species, and indeed, we identified 84 full length sequences including 22 novel sequences. Among the novel sequences, 8 sequences were previously unannotated and 14 sequences came from the partial correction of already available sequences. The CARP VIII and X sequences were found in all the available vertebrate genomes. However, CARP XI sequences were limited to mammals, frogs, lizards, and lobe-finned fish. Our bioinformatic analysis suggested that the CA11 gene emerged through gene duplication from CA10 either after the divergence of the fish and tetrapod lineages, or from the whole-genome duplication in early vertebrates. The CA11 gene was not found in the two bird genomes available for study I, and not in the four bird genomes at the time of writing of this thesis, so I assume it was lost in the bird lineage. Interestingly, the fish CARP X-like sequences formed two sub-groups of their own, suggesting that they appeared by CA10 gene duplication in the ray-finned fish lineage. The unexpected discovery of the study was the identification of CARP VIII-like sequences from deuterostomes. The finding of CARP VIII like sequences in deuterostomes suggested that these sequences might have originated after the separation of Protostomia and Deuterostomia lineages. After the publication of the first study of this thesis (I), a CARP VIII-like protein has been reported in the pearl oyster, Pinctada fucata (Miyamoto, 2012), and a mollusc, i.e. a protostome. More recently, three additional CARP VIII-like protostomal sequences have been added to online databases, namely two from other molluscs (Lottia gigantea, giant owl limpet, UniProt V4C962_LOTGI, January 2014); and Aplysia californica, California sea hare, RefSeq XP_005107445.1, July 2013) and one from an annelid worm (Capitella teleta, a bristle worm, GenBank ELT96119.1, January 2013). Their high sequence similarity, 50% to 58% identity with human CARP VIII (compared to 30% to 34% identity with human CARP X) proves a close relationship, and all of these sequences miss one or two of the zinc-binding histidines. Therefore, I need to reject our previous hypothesis on the origin of CARP VIII in

48 deuterostomes, as it appears in molluscs and annelids as well. Its origin must date back to protostomes, but it remains unexplained why it is not found in arthropods or nematodes. Miyamoto suggests (Miyamoto, 2012) that the mollusc CARPs would have originated independently from deuterostome CARP VIII, but other plausible explanations might include lineage-specific gene losses or horizontal gene transfer between an early deuterostome and an ancestor of molluscs and annelids. I have prepared a further phylogenetic tree for this thesis to illustrate the phylogenetic position of the invertebrate CARPs relative to vertebrate CARPs. As seen in figure 6 below, the genes coding for Drosophila and C. elegans CARPs are most similar to the CARP X/XI group of vertebrates, and need to be considered as many-to-many orthologs (co-orthologs) of CA10 and CA11 in human, whereas we have not discovered multiple CA8–like sequences in any genome yet.

Figure 6. A phylogenetic tree made of the coding sequences of 26 CARP genes. Neisseria gonorrhoeae alpha CA served as an outgroup. In preparation, a protein alignment was created using Clustal Omega (Sievers et al., 2011). The amino acid positions were used as a guide from which a codon alignment was made using the Pal2Nal program (Suyama et al., 2006). Finally a phylogenetic tree based on the generalized time-Reversible (GTR) model was computed in the Mr. Bayes program (Ronquist et al., 2012). After 10 million generations the average standard deviation of split frequencies was 0.008. The numbers at the nodes represent the posterior probabilities of each node.

The ancestor of CA8 remains an open question that awaits further study, even though CA10-like genes would be the obvious candidate. Despite their presence in all major groups of bilaterian animals, CARP X-like sequences are seemingly missing from some species. For example, our studies have not found them in the genomes of Ciona intestinalis or Ciona savignyi. The question whether this proves gene loss in the evolution of Ciona or whether these genes have escaped sequencing can only be answered when genomes with higher coverage become available.

6.1.2 CARPs are mainly expressed in the CNS (I)

In the past, RT-PCR and WB showed the presence of CARPs in the brain and few other tissues, often with conflicting results (Akisawa et al., 2003; Hirasawa et al., 2007; Hirota et al., 2003; Jiao et al., 2005; Taniuchi et al., 2002a). Hence, the purpose of the study (I) was to comprehensively analyze the expression pattern of the CARP genes in mouse, which is an important mammalian model organism. Previous studies on the expression of CA8 gene were done in several human tissues and showed that the CA8 is mainly expressed in the brain, while additionally expressed in the lung, liver, heart, skeletal muscle and low level of expression in the kidney (Okamoto et al., 2001). In the present study, the expression of Car8 mRNA was predominantly high in the cerebellum followed by liver and lung. Low to faint signals for Car8 mRNA were also seen in almost all the tissues we studied. Our findings on Car8 expression pattern agreed quite well with Okamoto´s results (Okamoto et al., 2001). In this study, immunohistochemistry showed strong signal for the presence of the CARP VIII protein in cerebellum and cerebrum which agree with the results of other previous studies (Hirota et al., 2003; Jiao et al., 2005; Lakkis et al., 1997b; Taniuchi et al., 2002a; Taniuchi et al., 2002b). Surprisingly, strong localized signals were also observed in the stomach, Fig 9 D (I) compared to the levels of mRNA in the stomach. We believe that the difference between the expression of CARP VIII protein and mRNA in the stomach might be due to the region of the stomach used for the analysis of Car8 mRNA as the expression of CARP VIII protein is localized in the stomach. The predominant expression of CARP VIII in the brain and high level of expression in several other tissues suggesting that CARP VIII might play a physiological role in these tissues. Earlier studies showed moderate to low levels of CA10 mRNA in the human brain, testis, salivary glands, kidney, pancreas, liver and testis (Okamoto et al., 2001). At protein level, human cerebellar PCs displayed a weak signal for CARP X (Nishimori et al., 2003; Taniuchi et al., 2002a). In our study, the strong signal for Car10 mRNA was seen in the cerebellum, parietal cortex and frontal cortex and a faint signal was seen in the midbrain and eye. The immunohistochemical study showed a localized signal for CARP X in the respiratory epithelium of the lung and a weak signal in the stomach and cerebral capillaries.

50 In the present study, CARP XI expression analyses showed a widespread distribution of Car11 mRNA with an exceptionally high level in the frontal cortex, parietal cortex, cerebellum and midbrain. Previously, high levels of CA11 mRNA was seen in all parts of the human brain and low levels in several other tissues, which included the kidney, liver, salivary glands, lung, skeletal muscle, pancreas and liver (Fujikawa-Adachi et al., 1999). The results of our RT-qPCR showed Car11 mRNA in the colon, kidney, ovary, heart and lung, which is in agreement with the results of the above study. The presence of high levels of CARP XI in the human and mouse CNS suggests a role for CARP XI in mammals. In the present study, the signal for CARP XI in the mouse brain was less intense in comparison with CARP VIII staining, which agrees with the results of a previous study on the human brain (Taniuchi et al., 2002a).

6.1.3 Sequence and structural analysis of CARP VIII (II)

There are several pieces of evidence showing that CARP VIII is an important protein in various species: First, our results showed that CARP VIII is highly conserved across the species, which can be considered one sign for an evolutionally important role (I). Second, interaction of CARP VIII with ITPR1, a Ca2+ channel protein is well known (Hirota et al., 2003). Third, defects in the CARP VIII gene lead to ataxia in mouse and human (Jiao et al., 2005; Kaya et al., 2011; Turkmen et al., 2009). The crystal structure of CARP VIII has been resolved and the basis of its inactivity has been reported (Picaud et al., 2009). Based on the above information, we have analyzed CARP VIII at structural level to get some novel insights into its possible functions (II, III). Comparative analysis of human CARP VIII (hCARP VIII) sequence with hCAs (hCAII, and hCA XIII) showed the presence of Glu stretch at N-terminal region of CARP VIII. The analysis also revealed the conservation of aa residues at the active site except His94 and Gln92, which were replaced by Arg117 and Glu115 in CARP VIII respectively. The Glu residues which are encoded by GAG repeats are found in all the mammalian CARP VIII proteins. It is well known that the expansion mutation in trinucleotide repeats lead to neurodegenerative disorders in humans (Plassart and Fontaine, 1994). It is speculated that the expansion mutation of GAG repeats in CA8 gene might lead to neurological disorders in humans. Similarly, it has been reported that the transcriptional activators GAL4, GCN4, and Herpesvirus VP16 contain a glutamic acid-rich region (Melcher, 2000; Tavernarakis and Thireos, 1995; Triezenberg et al., 1988). Therefore, the presence of a glutamic acid stretch in CARP VIII may be involved in the regulation of transcription (Miyaji et al., 2003) (II). Analysis of conserved residues in hCARP VIII compared with hCAII from a number of species showed a very high conservation of residues on the surface CARP VIII. The conservation of residues on the surface of protein suggests that these residues might be involved in the interaction with other proteins.

A homozygous missense mutation in the CA8 gene, resulting in substitution of Ser 100 by a Pro residue, was found in members of Iraqi family (Turkmen et al., 2009). The affected members of the family exhibited a quadrupedal gait with mild mental retardation. The in-vitro studies showed that the S100P aa change caused proteasomal mediated degradation of CARP VIII protein due to misfolding which lead to decreased levels of CARP VIII protein. This is a common mechanism for loss of function mutations associated with human disease (Wang and Moult, 2001). We studied the structural basis of misfolding in the S100P mutant protein showing the location of Ser 100 at the end of β strand β6. The substitution of Ser with Pro causes a rigid bend in the peptide backbone and thus preventing the β6 and or the β6/ β7 loop from assuming the confirmation similar to WT protein. The misfolding of CARP VIII would then lead to the proteasomal mediated degradation of the protein in the members of Iraqi family with a mutation in CA8 gene.

6.1.4 Zebrafish CARP VIII and ITPR1 sequences are coevolved (III)

CARP VIII and ITPR1 are co-expressed in the cerebellar PCs and are known to interact with each other (Hirota et al., 2003). The coevolution (a change in the gene product of one gene reflects a change in the gene product of another gene to retain the functional interaction) analysis of 31 vertebrate CARP VIII and ITPR1 sequences, which included 6 ITPR1a/ ITPR1b sequences from fish, showed a strong correlation with ITPR1a suggesting that ITPR1a and CARP VIII evolved together. The measurement of itpr1a and ca8 mRNAs indicated a similar expression pattern during the embryonic development, supporting the co-evolution of both genes and their functional association. In our study, the abnormal development of zebrafish larvae might be due to knockdown of ca8 gene leading to an improper modulation of ITPR1 function in Ca2+ signaling. Similarly, gait disorder and ataxia in mouse and human with a mutation in Car8 or CA8 gene may be due to improper interaction of CARP VIII with ITPR1. The mutations in ITPR1 gene also lead to ataxia in mice and SCA 15 in humans (van de Leemput et al., 2007).

6.1.5 The ca8 morphants displayed abnormal swim pattern (III)

CARP VIII is abundantly expressed in the human and mouse brain (Hirota et al., 2003; Jiao et al., 2005; Taniuchi et al., 2002a; Taniuchi et al., 2002b). Studies in the past showed that a spontaneously occurring mutation in CA8 gene leads to ataxia and mental retardation in humans (Kaya et al., 2011; Turkmen et al., 2009). However, no studies on the role of CARP VIII during embryonic development are available. The genes involved in the neurodegenerative disorders play a crucial role in the development of the brain early in embryogenesis and the molecular basis of brain development is similar in all the vertebrates. Lately, zebrafish has emerged as an

52 attractive model to study development in vertebrates, due to easy tractability and common biological relevance (Carradice and Lieschke, 2008; Lohi et al., 2013). In this study, we knocked down the ca8 gene to understand its consequences on the zebrafish brain at the ultrastructural level and behavioral consequences in morphant zebrafish. Zebrafish CARP VIII sequence analysis showed 84% similarity with hCARP VIII sequence in the core domain. Developmental expression analysis of ca8 gene indicated the presence of a high level of ca8 mRNA at 0 hpf, suggesting that the mRNA is of maternal origin and is required very early in development. Few reports are available on the developmental expression of the CARP VIII gene in mouse and human (Lakkis et al., 1997a; Taniuchi et al., 2002a; Taniuchi et al., 2002b). In one study, the expression of Car8 mRNA was studied by in-situ hybridization in mouse, which showed a positive signal at 9.5 days of gestation, while the expression data before 9.5 days of gestation was not available (Lakkis et al., 1997a). In the second study, the developmental expression of Car8 was investigated starting at 7 days of gestation until 17 days after fertilization (Taniuchi et al., 2002b). The RT- PCR and NB analyses suggested slightly conflicting results with positive signals at 11 and 15 days of gestation respectively. Similarly, the developmental expression studies in human showed CA8 expression at 84 days of gestation with some discrepant findings between the results of NB and immunohistochemistry (Taniuchi et al., 2002a). The reinvestigation of CARP VIII gene expression is needed in another mammalian model to get a better picture of CARP VIII gene expression during development. The expression pattern of zebrafish ca8 gene was studied using RT-qPCR in a panel of adult zebrafish tissues. Predominantly, expression of the ca8 gene was seen in the brain, with moderate to low levels of expression in several other tissues (III). At the protein level, expression of CARP VIII was seen in cerebellar PCs (III). The expression pattern of the ca8 gene in this study is fairly similar to the expression pattern in mouse (I) and human (Taniuchi et al., 2002a). The expression pattern of CARP VIII gene in the zebrafish brain – similar to mouse and human - further indicated a possible role for CA8 in vertebrates. The presence of similarities between the zebrafish, mouse, and human CARP VIII gene at both sequence and expression levels prompted us to silence the ca8 gene in zebrafish larvae to study if we could observe a phenotype similar to mouse and human patients with a defect in the CARP VIII gene. The morphant zebrafish injected with CA8-MOs (125 µM) exhibited several phenotypic defects (I). The injections of two different sets of MOs resulted in the similar phenotypic defects in the morphant embryos and injection of control MOs showed phenotypes similar to WT embryos. The injection of p53 MOs could not improve the defective phenotype of the morphant embryos, suggesting the phenotype obtained was due to the knockdown effect of MOs. The efficacy of the translation blocking MOs was further confirmed by a WB of lysates from 3 dpf embryos and analyzing the length of ca8 mRNA in MO2 morphant zebrafish.

Previous studies of the cerebellum of wdl mice showed defects in the morphology of the synapse, suggesting a critical role for CARP VIII in formation and/or maintenance of synapse and its excitatory function in the cerebellum (Hirasawa et al., 2007). Another study showed a loss in the cerebellar volume proportional to the age in members of Saudi family (Kaya et al., 2011). In the present study, ca8 morphant fish exhibited abnormal development of the head at 1 dpf stage, the time point at which differentiation of neurons occurs. Histological studies on 5 dpf morphant fish revealed gross morphological changes in the cerebellar region. In addition, TUNEL assay indicated apoptosis of neurons in the cerebellum and electron microscopy also showed increased neuronal cell death. The changes observed in the cerebellum of 5 dpf morphant zebrafish may be similar to the changes found in the cerebellum of human patients (Kaya et al., 2011). In the present study, we found that an increase in the concentration of ca8-MOs lead to more severe phenotype in the morphant fish. More importantly, the morphant fish injected with lower concentration of ca8-MOs, with a phenotypic appearance similar to the WT fish, displayed abnormal swim pattern. Taken together, the ca8 morphant fish mimicked the phenotype observed in wdl mouse and human patients who had a mutation in CA8 gene (Jiao et al., 2005; Kaya et al., 2011; Turkmen et al., 2009). Taken together, our studies suggest that down regulation of CARP VIII expression might disturb the Ca2+ ion channel in the endoplasmic reticulum by increasing the sensitivity of IP3 to ITPR1 in the absence of CARP VIII protein. Thus releasing large amount of Ca2+ from intracellular reserves activating and deactivating many genes including the genes involved in apoptotic pathway leading to neuronal cell death and ataxia in the present study and human patients and mouse with a mutation in CARP VIII gene. Indeed, analysis of expression profile of genes from the mouse with a spontaneous mutation in CARP VIII gene showed dysregulation of many genes which play important roles in the brain

6.1.6 Zebrafish ca10a and ca10b genes are widely expressed (IV)

Similar to CARP VIII, the sequences of CARP X and XI are highly conserved across species. Unlike CARP VIII, very few reports are available on CARP X and CARP XI, which makes these two proteins even more attractive research targets. First, we performed bioinformatic analysis on CARP Xa and CARP Xb sequences to understand their evolutionary relationship and conservation. In the past, some studies suggested the presence of signal peptides in CARP X and CARP XI (Bellingham et al., 1998; Fujikawa-Adachi et al., 1999; Okamoto et al., 2001), and later on another study suggested cytoplasmic localization for CARP X and CARP XI based on the results of phylogeny and histochemical staining (Taniuchi et al., 2002b). To resolve the earlier contradictory findings we used bioinformatic tools to look at the features, such as disulfides and N-glycosylation motifs that have functional significance in secreted proteins. Our sequence analysis

54 revealed the presence of a signal peptide in all the CARP X and XI sequences we analyzed (Fig 4, IV). Similarly, the sequence analyses revealed a pair of cysteines in the same location where all the active extracellular CA isoforms have a disulfide bridge. In addition, we found two conserved N-glycosylation motifs in all the sequences analyzed in the study (Fig 4, IV). The above finding of our study strongly suggests that the CARP Xa and Xb sequences and their vertebrate and invertebrate orthologs are secretory proteins. Expression analysis suggested that the ca10a and ca10b mRNAs are present throughout the development of zebrafish. The highest level of ca10a mRNA was found after 96 hpf. The presence of high levels ca10b mRNA at 0 hpf suggested that it is of maternal origin. Similarly, the presence of a high level of ca10b mRNA between 48 hpf and 168 hpf suggests a possible role in the development of zebrafish. In the past, developmental expression studies of CARP X and CARP XI genes were done in mouse whole embryos and in the human fetal brain (Taniuchi et al., 2002a; Taniuchi et al., 2002b). In mouse embryos, the expression of Car10 and Car11 genes was studied only at four different stages of development (E7, E11, E15 and E17). The presence of the Car10 gene was observed in the middle of gestation (E15) and Car11 appeared early in the development (Taniuchi et al., 2002b). In the human fetal brain, expression of CARP X and CARP XI was studied using immunohistochemistry at five different stages of development (Taniuchi et al., 2002a). In this study too, the signal for CARP X appeared late during the development (121st day of gestation) compared with the signal for CARP XI, which appeared at 84th and 95th day of gestation. The results of these studies are in agreement with the results of the present study in the zebrafish model. Previous studies showed that the CARP X and CARP XI genes are predominantly expressed in the human and mouse CNS (Bellingham et al., 1998; Okamoto et al., 2001; Taniuchi et al., 2002a; Taniuchi et al., 2002b). In the present study, the expression pattern of ca10a and ca10b genes in adult zebrafish tissues showed that the ca10a gene is predominantly expressed in the brain and also exhibited high level of ca10b gene in the brain. Thus these results agree well with the results of above studies. Surprisingly, the highest level of ca10b signal was detected in the zebrafish ovary, which is in contrast with the expression pattern in human and mouse. Interestingly, D. melanogaster contains two CARP sequences. CARP A, which is similar to CARP X, is expressed in the brain and CARP B, which is similar to CARP XI, is predominantly expressed in the reproductive tissues followed by CNS (Ortutay et al., 2010, Tolvanen et al unpublished results). Similarly, C. elegans contains two copies of CARP X like genes known as cah-1 and cah-2 which are expressed in several types of neurons (Spencer et al., 2011). Taken together, the results of the expression patterns of ca10a and ca10b genes suggest that these genes may play an important but yet unknown roles for these genes in the brain.

6.1.7 Ataxia and apoptosis in ca10a and ca10b morphants (IV)

Recent study showed an upregulation of CARP XI and altered localization in cultured neuronal cells expressing the mutant ataxin 3 (Hsieh et al., 2013). Further analysis of expression of CARP XI in SCA 3 and in MJD mouse revealed results similar to neuronal cells expressing mutant ataxin 3. It was suggested that CARP XI might interact with ITPR1 and control the release of Ca2+ similar to CARP VIII (Chen et al., 2008; Hsieh et al., 2013). Taken together, the results of the present study suggest that the ca10a and ca10b genes are required for the normal development of the brain during embryogenesis and the suppression of these genes lead to ataxic phenotype in the zebrafish. However, more studies are needed to understand the precise function of the CARP X and CARP XI proteins.

56 7 Summary and future directions

In the present work, I have studied the distribution and phylogenesis of CARP VIII, CARP X, and CARP XI sequences from all the available databases and studied the expression of all the three CARPs both at the gene and protein levels in mouse. I also studied the functional role of CARP VIII, CARP X and CARP XI proteins in zebrafish using antisense morpholinos to silence the ca8, ca10a and ca10b genes. Bioinformatic analysis showed a very high conservation of CARP VIII, X and XI sequences across the species studied. The study revealed unusually high similarity within CARP VIII and X groups, even between invertebrate and vertebrate sequences. The high conservation of CARPs suggests conserved functions across the vertebrate species. Results of the phylogenetic analysis further revealed that duplication of CA10 gene followed different paths in the fish and tetrapod lineages. The expression analyses of CARP genes suggested that the Car8 gene is expressed in a wide variety of tissues in the mouse with a predominantly high level of expression in the cerebellum. The strongest expression of Car10 and Car11 genes was seen in the CNS. The expression patterns of CARP genes suggest that these proteins may be involved in the development of the nervous system and nerve system functions, such as motor coordination. In addition, they may have still unknown physiological roles in other tissues. In zebrafish, CARP VIII was expressed in the brain and in several other tissues, which is similar to the expression pattern in mouse and human tissues. The presence of CA8 gene product in 0 hpf embryos suggested that the gene is of maternal origin. Knockdown of the CA8 gene led to abnormal swim pattern in morphant fish with a reduction in cerebellar volume. The morphant zebrafish displayed ataxic movement similar to wdl mice and human patients. The study provides evidence that CARP VIII is required in early development in zebrafish, especially for the normal development of the brain. Studies carried out on CARP X and CARP XI proteins showed that they are highly expressed in the CNS of the zebrafish. Data on the developmental expression pattern of ca10a and ca10b genes in zebrafish are novel and suggest important but unknown roles for these genes in embryonic development. This is the first study to demonstrate that silencing of ca10a or ca10b gene leads to abnormal embryonic development. Knockdown of ca10a and ca10b genes results in an abnormal swim pattern in the morphant zebrafish and suggests that these genes might regulate motor coordination. Further experiments are needed to precisely define the role of CARP X and CARP XI proteins in mammals.

It is well established that the CARP VIII is predominantly expressed in cerebellar Purkinje cells in human, mouse and zebrafish and it also interacts with ITPR1 receptor in these cells. Association of CARP VIII with motor coordination function and in neurodevelopmental disorder has been demonstrated in human and mouse. The association of CARP VIII has been also studied in human cancers and cancer cell lines. However, the precise role of CARP VIII still remains elusive. The progress made so far on CARP VIII research will be helpful to design future course of research to define the precise role of CARP VIII both in motor coordination function and human cancers. In fact, in our laboratory work is in progress in cultured cells and in human cancers to study the mechanism by which CARP VIII exerts its functions in both cancers and in neurodevelopmental disorder. Studies done in the past showed that similar to CARP VIII expression CARP X and CARP XI are also predominantly expressed in the CNS. In the present study the experiments in zebrafish showed that the CARP X and CARP XI play a crucial role during embryonic development and play a role in motor coordination function. However, unlike CARP VIII very little is known about their mode of action and their precise roles. The research so far showed that the CARPs function by interacting with other proteins. It will be interesting to find binding partners for CARP X and CARP XI using different molecular techniques. More information and better insights into the role of CARP X and CARP XI can be obtained by knocking down Car10 and Car11 genes individually (single knockout) and together (double knockout) in a mouse model. The CA10 gene is known to contain CCG repeats and any expansion mutation in these repeats might lead to a neurodegenerative disease in humans. It will be interesting to do the large scale screening of the patients with unknown neurological disorders for the expansion mutations in CA10 gene. To get better insights into the biological role of CAPR X and CARP XI in human cancers we plan to use cell culture techniques and analyze human cancer tissues.

58 Acknowledgements

This research was carried out at the Institute of Biomedical Technology (IBT), now BioMediTech and Department of Anatomy, School of Medicine, University of Tampere, Finland in the years 2010-14. I am deeply indebted to my supervisors, Prof. Mauno Vihinen, PhD., and Prof Seppo Parkkila, MD., PhD. To Prof. Mauno, for accepting me for master’s study in his department and giving me an opportunity to study bioinformatics. Perhaps most importantly, I am thankful for him sharing his enthusiasm for research, further encouraging me, accepting me as a PhD candidate, for his positive feedback, and for giving me help throughout the program, without which this thesis would not have been possible. I will always be grateful to Prof. Seppo Parkkila first, for providing me a position in his group and then giving all the freedom I could ever ask for, and showing faith in me. I consider myself privileged to work in his group and it was a real turning point in my research carrier. Biological research has always been close to my heart and working with Seppo made me enjoy biological research more than ever. I thank him for investing in me, and making sure no opportunity for a challenge, competition, scholarship, presentation/poster, or conference passed by. Beyond science, you have mentored my growth as a person throughout these years, and have encouraged me to never fall short of my dreams. That is a tremendous gift, and I owe you infinite gratitude. I am grateful for his guidance and encouragement during these years, as well as his patience and flexibility as I struggled to combine work and family life successfully. I express my sincere thanks to Assoc. Prof. Martti Tolvanen PhD, Bioinformatics Unit, University of Tampere and presently at University of Turku. Martti is my teacher, colleague and friend and introduced me to research these fascinating proteins, CARPs. Without Martti’s contribution to the CARP project, my thesis would be incomplete. His endless discussions, be it one to one or in the group meetings, and his practical guidance on the thesis project, were of immense value. His collaboration in the research projects, including CARPs, have produced several high impact factor publications apart from my PhD thesis. His patience and generosity be it in research or outside of research, has taught me many things that will go a long way in life. If I had a problem, however big or small, if I turned to Martti for help, he would help me every single time. Apart from research, I enjoyed my time with him on numerous occasions. Be it in Tampere, or in conferences such as Florence, Italy and Antalya, Turkey, just sipping beer, watching movies in theater, having food (we both enjoy all kinds of foods in the world), and it was a

59 pleasure cooking and sharing Indian food with Martti. Thank you very much Martti for your kindness and help, which made all these years enjoyable and the memories will always remain with me. Sometimes you become friends with someone and feel that you have always known that person. It was so nice to meet Harlan Barker (M.Sc. Bioinformatics) through peer-tutoring when he came from the US in 2011 for a bioinformatics program at the University of Tampere. Later, it was a great pleasure to have him sitting in the same office next to me as a colleague. Indeed, I owe many thanks to Harlan for his help in many things in and outside of research. It was great fun to discuss many topics ranging from research to as wide as politics, movies and life. We had great time together in the lab chatting, meeting over food and beer, watching movies in the theater thanks for that. His contribution to my PhD thesis as a co- author and his invaluable help with the English language corrections in the PhD thesis manuscripts are greatly appreciated. I wish to thank my thesis committee members, Docent Dr. Csaba Ortutay, PhD, Bioinformatics group, University of Tampere and Prof. Heikki Kainulainen MD, PhD, Department of Biology of Physical Activity University of Jyväskylä for their timely assessment and annual reports for TGPBB. I would also like to thank Dr. Ortutay for his collaboration with the PhD thesis project and for the bioinformatic analysis work that lead to many good publications together. I would like to acknowledge the director of the Institute of Biomedical Technology and the Director of BioMediTech, Dr. Hannu Hanhijärvi, DDS, Ph.D., for providing me the research facilities for this study. My thanks are also due to the administrative staff of the institute and Medical School, especially, Riitta Aallos, Marjatta Viilo, Jaana Anttila-Salmensivu, Merja Koivula, Östring Anne- Marja, Mira Pihlström, I express my gratitude to Sari Luokkala, the computer administrator B-building, for all the help with my computers during the course of my research which was so invaluable. I am grateful to Jukka Lehtiniemi for the help with movement pattern videos for research articles, and for my information at IBT web page. My special thanks are also due to Dr. Henna Mattila, PhD, and the Tampere Graduate Program in Biomedicine and Biotechnology (TGPBB), for supporting me and for the courses that were of immense help to me to grow as a researcher in particular, and as a person in general, and for the financial support for attending the international conferences. I would also like to thank Docent Vesa Hytönen, PhD for his help with experiments and for the discussions related to my research and my PhD project and his guidance. Dr. Hytönen’s enthusiastic attitude towards science and his endless thirst for knowledge have been very inspiring and encouraging. I greatly appreciate and thank Prof. Abdul Waheed, PhD, Saint Louis University School of Medicine, USA, for the discussion and guidance on my thesis project and for sharing his wisdom and enthusiasm in several conference meetings and during his visits to School of Medicine, Tampere. I am thankful to Dr. Jarko Valjakka and Janette Hinkka from FinMedi1 for their kindness and all the help every time I went to their office if I needed anything. I want to express my deepest

60 gratitude to all my colleagues at IMT and to thank the former and current attendants for always being so helpful and keeping things running. The official reviewers of my thesis, Prof. Todd Alexander, MD, PhD, FRCP, Associate Professor, University of Alberta, Canada and Prof. Matti Airaksinen, MD, PhD, Professor of Neuroscience, University of Helsinki, Finland are warmly thanked for their constructive criticism and feedback that lead to an improvement in the quality of my thesis. It is an honor to have Prof. Erik Pettersen, University of Oslo, Norway as the opponent of my thesis and I am grateful to him for accepting the invitation. I would like to thank my collaborators and co-authors, without their contribution, this thesis would not have been completed. Most of my thesis work is based on functional studies of CARPs using morpholino (MO) knockdown technology in a zebrafish model. I am grateful to Prof. Mika Rämet MD, PhD, Experimental Immunology group, BioMediTech, Unversity of Tampere, for the collaboration with knockdown experiments in zebrafish that produced some wonderful results and manuscripts. I thank Prof. Rämet for extending me all the help and freedom I needed throughout the work. I appreciate the critical review of the manuscripts, input, and the discussions that were of immense help for the thesis and for me personally. I am grateful to Dr. Mataleena Parikka, DDE, PhD, for teaching and helping me with basics of zebrafish experiments, MO injections, handling and manipulation of zebrafish embryos, and for being there for me every time I needed her help and for her critical comments and review of the manuscripts. I appreciate and thank Sanna- Kaisa E. Harjula, M.Sc., for her immense help especially during the MO injections and also with using the microscope and taking the swim pattern videos. I greatly appreciate the help of Leena Makinen for doing morpholino injections for knockdown of CARP genes and arranging the embryos and adult fish for my experiments within one day of request. I would thank Hannaleena Piipo for all the arrangements and help with zebrafish embryos and with MO knockdown experiments during the research. I thank Matilda Martikainen and Kaisa Oksanen for technical assistance during the MO knockdown experiments. I would also like to thank Docent Hannu Turpeinen, PhD for the help and discussion regarding the rescue of morphant fish. Thanks are also due to Annemari Uusimäki for her technical help and for the cDNAs from zebrafish adult tissues. I appreciate Dr. Kaisa Teittinen, PhD for the help with pDNA3.1 for the synthesis of CARP mRNA for the experiments. I would like to thank Dr. Eija Jokitalo PhD, Electron Microscopy Unit, Institute of Biotechnology, University of Helsinki for her collaboration with ca8 morphants electron microscope studies, which produced wonderful results and lead to a good publication. I consider myself lucky to have wonderful colleagues and friends in the laboratory and at the IBT. It was pleasure working with Dr. Peiwen Pan PhD, Marianne Kuuslahti, Aulikki Lehmus, Dr. Leo Syrjänen MD, Dr. Heini Kallio, PhD, Maarit Patrikainen M.Sc, and Reza Zolfaghari Emameh M.Sc, Saku Peltari, Prajwol Manandhar, Lydia Isokangas and Rawnak Jahan Hoque. I would especially like to

61 thank Leo and Heini for their friendship and help in the lab with the experimental work that were of immense help during my initial days in the CA group. In the later part of my graduate program, it was great pleasure to work with Maarit on CA related projects. Together we have produced a lot of important scientific data that is in the process of publication in high impact factor journals. My special thanks go to Peiwen for her friendship and the wonderful time we had in the lab, be it doing experiments or discussion about research. Apart from research, I appreciate her help and guidance in my personal life and several get togethers we had with Dr. Yuemei Fan, PhD for Chinese and Indian food and long discussions about life and other things in general, but also for the many interesting discussions we’ve had in appreciation of our different cultures and languages thank you Yuemei and Peiwen. Marianne has my appreciation and deserves a huge thanks for all the technical help and assistance with the experiments and discussions, without which my thesis would be incomplete. My special thanks are also due to Aulikki Lehmus for all the help in the lab and her expert technical assistance and guidance in the laboratory and for your kindness. I would like to thank Dr. Pasi Pennanen, PhD for his help from time to time in the department and invaluable help with qPCR calculations every single time I needed. It was indeed a pleasure to work alongside with Dr. Heimo Syvälä, PhD, Sami Purmonen, M.Sc, Prof. Teemu Ylikomi, MD, PhD, Ninni Ikkonen, Arja Ahola, and Ilka Mäkinen. I would like to thank Arja specially for giving me day-to-day help in the department whenever I needed it and it was always a pleasure talking to her, who is ever smiling and enthusiastic and who would always lift my mood. My thanks also go to the past CA group members Alejandra, Mikä, and Fatemeh. I am thankful to my friends, especially FCA. Naveen Raj from India for being with me in all the situations, Dr. Peter Carvalho PhD (Org Chem) from Germany for being a great buddy and helping me with many things and spending endless hours talking about research and life in the GCH Hostel, Mumbai, India that helped me learn many things and my thanks to Dr. Hormudz Mulla PhD (Org Chem), Arizona University, Head R&D at Sudarshan Chemicals Limited's Pigments Pune, India for the friendship. I do not have words to express my gratitude to Prof. Iqbal Hamza PhD, Dept. of Animal & Avian Sciences, University of Maryland, USA, your friendship and association had a great influence on me ever since we became friends at The Institute of Science, Mumbai and it will always have the same effect. Thanks for teaching me the things that nobody would teach and one cannot learn at schools, I am indebted to you for the same. I would like to thank my friends and colleagues in Tampere, especially the late Ayodeji Olatubosun, Gabriel Teku, Mesue Kolle, Sreevani Kotha and Rishi Mohanta for being with me when I needed them and helping me academically as well as personally and making my stay in Tampere pleasant. My special thanks go to Chandrasekhar Challagonda and Vijender Reddy for their generous help in the initial period of my stay in Tampere and for their friendship. My sincere thanks go to our family friends Imre and Daniela Balog for their friendship and for their generosity and for being there for us for any help we need.

62 This study was financially supported by the Fimlabs Research Fund, the Pirkanmaa District Hospital, Medical Research Fund of Tampere University Hospital, The Sigrid Juselius Foundation, Academy of Finland, Tampere University Foundation, the Scientific Foundation of the City of Tampere, and Jane and Aatos Erkko Foundation. I thank my late parents for the love and support and for inculcating good values in me without which I would never have reached where I am today. I am grateful to Amma, Bapu and Akkalu, Geetha and Srilatha and their families for giving me unconditional love and support, which means a lot to me. I thank my girlfriend Sanji, for being there for me and for her love and support, which will always mean a lot to me and I thank my son Atharva, whom I love very much, for being my source of strength and inspiration and my son Shiv who is a source of joy and motivation and to both my sons for providing a reason to live my life. Indeed, I am grateful to the God for giving me strength to carry through the difficult times and for being kind to me.

Tampere, October 2014

Ashok Aspatwar

References

Akisawa, Y., Nishimori, I., Taniuchi, K., Okamoto, N., Takeuchi, T., Sonobe, H., Ohtsuki, Y., and Onishi, S. (2003). Expression of carbonic anhydrase-related protein CA-RP VIII in non-small cell lung cancer. Virchows Arch 442, 66-70. Altschul, S.F., Madden, T.L., Schaffer, A.A., Zhang, J., Zhang, Z., Miller, W., and Lipman, D.J. (1997). Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 25, 3389-3402. Andersen, J.N., Jansen, P.G., Echwald, S.M., Mortensen, O.H., Fukada, T., Del Vecchio, R., Tonks, N.K., and Moller, N.P. (2004). A genomic perspective on protein tyrosine phosphatases: gene structure, pseudogenes, and genetic disease linkage. FASEB J 18, 8-30. Barnea, G., Silvennoinen, O., Shaanan, B., Honegger, A.M., Canoll, P.D., D'Eustachio, P., Morse, B., Levy, J.B., Laforgia, S., Huebner, K., et al. (1993). Identification of a carbonic anhydrase-like domain in the extracellular region of RPTP gamma defines a new subfamily of receptor tyrosine phosphatases. Mol Cell Biol 13, 1497-1506. Bataller, L., Sabater, L., Saiz, A., Serra, C., Claramonte, B., and Graus, F. (2004). Carbonic anhydrase-related protein VIII: autoantigen in paraneoplastic cerebellar degeneration. Ann Neurol 56, 575-579. Behravan, G., Jonsson, B.H., and Lindskog, S. (1991). Fine tuning of the catalytic properties of human carbonic anhydrase II. Effects of varying active-site residue 200. Eur J Biochem 195, 393-396. Bellingham, J., Gregory-Evans, K., and Gregory-Evans, C.Y. (1998). Sequence and tissue expression of a novel human carbonic anhydrase-related protein, CARP-2, mapping to chromosome 19q13.3. Biochem Biophys Res Commun 253, 364-367. Birney, E., Clamp, M., and Durbin, R. (2004). GeneWise and Genomewise. Genome Res 14, 988-995. Bosanac, I., Alattia, J.R., Mal, T.K., Chan, J., Talarico, S., Tong, F.K., Tong, K.I., Yoshikawa, F., Furuichi, T., Iwai, M., et al. (2002). Structure of the inositol 1,4,5-trisphosphate receptor binding core in complex with its ligand. Nature 420, 696-700. Carradice, D., and Lieschke, G.J. (2008). Zebrafish in hematology: sushi or science? Blood 111, 3331-3342. Chen, X., Tang, T.S., Tu, H., Nelson, O., Pook, M., Hammer, R., Nukina, N., and Bezprozvanny, I. (2008). Deranged calcium signaling and neurodegeneration in spinocerebellar ataxia type 3. J Neurosci 28, 12713-12724. Elleby, B., Sjoblom, B., and Lindskog, S. (1999). Changing the efficiency and specificity of the esterase activity of human carbonic anhydrase II by site-specific mutagenesis. Eur J Biochem 262, 516-521. Elleby, B., Sjoblom, B., Tu, C., Silverman, D.N., and Lindskog, S. (2000). Enhancement of catalytic efficiency by the combination of site-specific mutations in a carbonic anhydrase- related protein. Eur J Biochem 267, 5908-5915.

64 Emanuelsson, O., Nielsen, H., Brunak, S., and von Heijne, G. (2000). Predicting subcellular localization of proteins based on their N-terminal amino acid sequence. J Mol Biol 300, 1005-1016. Fares, M.A., and McNally, D. (2006). CAPS: coevolution analysis using protein sequences. Bioinformatics 22, 2821-2822. Fujikawa-Adachi, K., Nishimori, I., Taguchi, T., Yuri, K., and Onishi, S. (1999). cDNA sequence, mRNA expression, and chromosomal localization of human carbonic anhydrase- related protein, CA-RP XI. Biochimica et biophysica acta 1431, 518-524. Gavrieli, Y., Sherman, Y., and Ben-Sasson, S.A. (1992). Identification of programmed cell death in situ via specific labeling of nuclear DNA fragmentation. J Cell Biol 119, 493-501. Han, M.V., and Zmasek, C.M. (2009). PhyloXML: XML for evolutionary biology and comparative genomics. BMC Bioinformatics 10, 356. Hewett-Emmett, D., 2000. Evolution and distribution of the carbonic anhydrase gene families. In: Chegwidden, W.R., Carter, N.D., Edwards, Y.H. (Eds.), Evolution and distribution of the carbonic anhydrase gene families. The Carbonic Anhydrases: New Horizons. Birkhauser Verlag, Boston, pp. 29–76. Hewett-Emmett, D., and Tashian, R.E. (1996). Functional diversity, conservation, and convergence in the evolution of the a-, b-, and g-carbonic anhydrase gene families. Mol Phylogenet Evol 5, 50-77. Higgins, D.G., Thompson, J.D., and Gibson, T.J. (1996). Using CLUSTAL for multiple sequence alignments. Methods Enzymol 266, 383-402. Hirasawa, M., Xu, X., Trask, R.B., Maddatu, T.P., Johnson, B.A., Naggert, J.K., Nishina, P.M., and Ikeda, A. (2007). Carbonic anhydrase related protein 8 mutation results in aberrant synaptic morphology and excitatory synaptic function in the cerebellum. Mol Cell Neurosci 35, 161-170. Hirota, J., Ando, H., Hamada, K., and Mikoshiba, K. (2003). Carbonic anhydrase-related protein is a novel binding protein for inositol 1,4,5-trisphosphate receptor type 1. Biochem J 372, 435-441. Hsieh, M., Chang, W.H., Hsu, C.F., Nishimori, I., Kuo, C.L., and Minakuchi, T. (2013). Altered expression of carbonic anhydrase-related protein XI in neuronal cells expressing mutant ataxin-3. Cerebellum 12:338–349 Ishihara, T., Takeuchi, T., Nishimori, I., Adachi, Y., Minakuchi, T., Fujita, J., Sonobe, H., Ohtsuki, Y., and Onishi, S. (2006). Carbonic anhydrase-related protein VIII increases invasiveness of non-small cell lung adenocarcinoma. Virchows Arch 448, 830-837. Jiao, Y., Yan, J., Zhao, Y., Donahue, L.R., Beamer, W.G., Li, X., Roe, B.A., Ledoux, M.S., and Gu, W. (2005). Carbonic anhydrase-related protein VIII deficiency is associated with a distinctive lifelong gait disorder in waddles mice. Genetics 171, 1239-1246. Kastury, K., Ohta, M., Lasota, J., Moir, D., Dorman, T., LaForgia, S., Druck, T., and Huebner, K. (1996). Structure of the human receptor tyrosine phosphatase gamma gene (PTPRG) and relation to the familial RCC t(3;8) chromosome translocation. Genomics 32, 225-235. Kato, K. (1990a). A Collection of cDNA Clones with Specific Expression Patterns in Mouse Brain. Eur J Neurosci 2, 704-711. Kato, K. (1990b). Sequence of a novel carbonic anhydrase-related polypeptide and its exclusive presence in Purkinje cells. FEBS Lett 271, 137-140. Kawaguchi, Y., Okamoto, T., Taniwaki, M., Aizawa, M., Inoue, M., Katayama, S., Kawakami, H., Nakamura, S., Nishimura, M., Akiguchi, I., et al. (1994). CAG expansions in a novel gene for Machado-Joseph disease at chromosome 14q32.1. Nat Genet 8, 221-228.

Kaya, N., Aldhalaan, H., Al-Younes, B., Colak, D., Shuaib, T., Al-Mohaileb, F., Al-Sugair, A., Nester, M., Al-Yamani, S., Al-Bakheet, A., et al. (2011). Phenotypical spectrum of cerebellar ataxia associated with a novel mutation in the CA8 gene, encoding carbonic anhydrase (CA) VIII. Am J Med Genet B Neuropsychiatr Genet 156, 826-834. Kent, W.J. (2002). BLAT--the BLAST-like alignment tool. Genome Res 12, 656-664. Kent, W.J., Sugnet, C.W., Furey, T.S., Roskin, K.M., Pringle, T.H., Zahler, A.M., and Haussler, D. (2002). The human genome browser at UCSC. Genome Res 12, 996-1006. Kiefer, L.L., Paterno, S.A. and Fierke, C.A. (1995). Hydrogen bond network in the metal binding site of carbonic anhydrase enhances zinc affinity and catalytic efficiency. J. Am. Chem.Soc 117, 6831-45837. Kilpinen, S., Autio, R., Ojala, K., Iljin, K., Bucher, E., Sara, H., Pisto, T., Saarela, M., Skotheim, R.I., Bjorkman, M., et al. (2008). Systematic bioinformatic analysis of expression levels of 17,330 human genes across 9,783 samples from 175 types of healthy and pathological tissues. Genome Biol 9, R139. Kim, D.S., Ross, S.E., Trimarchi, J.M., Aach, J., Greenberg, M.E., and Cepko, C.L. (2008). Identification of molecular markers of bipolar cells in the murine retina. The Journal of comparative neurology 507, 1795-1810. Kimmel, C.B., Ballard, W.W., Kimmel, S.R., Ullmann, B., and Schilling, T.F. (1995). Stages of embryonic development of the zebrafish. Developmental dynamics : an official publication of the American Association of Anatomists 203, 253-310. Kleiderlein, J.J., Nisson, P.E., Jessee, J., Li, W.B., Becker, K.G., Derby, M.L., Ross, C.A., and Margolis, R.L. (1998). CCG repeats in cDNAs from human brain. Hum Genet 103, 666-673. LaForgia, S., Morse, B., Levy, J., Barnea, G., et al. (1991). Receptor protein-tyrosine phosphatase gamma is a candidate tumor suppressor gene at human chromosome region 3p21. Proc Natl Acad Sci U S A 88, 5036-5040. Lakkis, M.M., Bergenhem, N.C., O'Shea, K.S., and Tashian, R.E. (1997a). Expression of the acatalytic carbonic anhydrase VIII gene, Car8, during mouse embryonic development. Histochem J 29, 135-141. Lakkis, M.M., O'Shea, K.S., and Tashian, R.E. (1997b). Differential expression of the carbonic anhydrase genes for CA VII (Car7) and CA-RP VIII (Car8) in mouse brain. J Histochem Cytochem 45, 657-662. Lamprianou, S., Vacaresse, N., Suzuki, Y., Meziane, H., Buxbaum, J.D., Schlessinger, J., and Harroch, S. (2006). Receptor protein tyrosine phosphatase gamma is a marker for pyramidal cells and sensory neurons in the nervous system and is not necessary for normal development. Mol Cell Biol 26, 5106-5119. Larkin, M.A., Blackshields, G., Brown, N.P., Chenna, R., McGettigan, P.A., McWilliam, H., Valentin, F., Wallace, I.M., Wilm, A., Lopez, R., et al. (2007). Clustal W and Clustal X version 2.0. Bioinformatics 23, 2947-2948. Levy, J.B., Canoll, P.D., Silvennoinen, O., Barnea, G., Morse, B., Honegger, A.M., Huang, J.T., Cannizzaro, L.A., Park, S.H., Druck, T., et al. (1993). The cloning of a receptor-type protein tyrosine phosphatase expressed in the central nervous system. J Biol Chem 268, 10573-10581. Lohi, O., Parikka, M., and Ramet, M. (2013). The zebrafish as a model for paediatric diseases. Acta paediatrica 102, 104-110. Lovejoy, D.A., Hewett-Emmett, D., Porter, C.A., Cepoi, D., Sheffield, A., Vale, W.W., and Tashian, R.E. (1998). Evolutionarily conserved, "acatalytic" carbonic anhydrase-related protein XI contains a sequence motif present in the neuropeptide sauvagine: the human CA-

66 RP XI gene (CA11) is embedded between the secretor gene cluster and the DBP gene at 19q13.3. Genomics 54, 484-493. Lu, S.H., Takeuchi, T., Fujita, J., Ishida, T., Akisawa, Y., Nishimori, I., Kohsaki, T., Onishi, S., Sonobe, H., and Ohtsuki, Y. (2004). Effect of carbonic anhydrase-related protein VIII expression on lung adenocarcinoma cell growth. Lung Cancer 44, 273-280. Lukacs, G.L., and Verkman, A.S. (2012). CFTR: folding, misfolding and correcting the DeltaF508 conformational defect. Trends Mol Med 18, 81-91. Maa, J.S., Rodriguez, J.F., and Esteban, M. (1990). Structural and functional characterization of a cell surface binding protein of vaccinia virus. J Biol Chem 265, 1569-1577. Melcher, K. (2000). The strength of acidic activation domains correlates with their affinity for both transcriptional and non-transcriptional proteins. J Mol Biol 301, 1097-1112. Mikoshiba, K. (2007). IP3 receptor/Ca2+ channel: from discovery to new signaling concepts. J Neurochem 102, 1426-1446. Mikoshiba, K., Okano, H., Miyawaki, A., Furuichi, T., and Ikenaka, K. (1995). Molecular genetic analyses of myelin deficiency and cerebellar ataxia. Prog Brain Res 105, 23-41. Miyaji E, Nishimori I, Taniuchi K, Takeuchi T, Ohtsuki Y, and Onishi, S. (2003). Overexpression of carbonic anhydrase-related protein VIII in human colorectal cancer. J Pathol. 201, 37-45. Miyaji, E., Nishimori, I., Taniuchi, K., Takeuchi, T., Ohtsuki, Y., and Onishi, S. (2003). Overexpression of carbonic anhydrase-related protein VIII in human colorectal cancer. J Pathol 201, 37-45. Miyamoto, H. (2012). Sequence of the pearl oyster carbonic anhydrase-related protein and its evolutionary implications. Biochem Genet 50, 269-276. Mori, S., Kou, I., Sato, H., Emi, M., Ito, H., Hosoi, T., and Ikegawa, S. (2009). Nucleotide variations in genes encoding carbonic anhydrase 8 and 10 associated with femoral bone mineral density in Japanese female with osteoporosis. J Bone Miner Metab 27, 213-216. Morimoto, K., Nishimori, I., Takeuchi, T., Kohsaki, T., Okamoto, N., Taguchi, T., Yunoki, S., Watanabe, R., Ohtsuki, Y., and Onishi, S. (2005). Overexpression of carbonic anhydrase- related protein XI promotes proliferation and invasion of gastrointestinal stromal tumors. Virchows Arch 447, 66-73. Nicholas, K.B., Nicholas H.B. Jr., and Deerfield, D.W. II (1997). GeneDoc: Analysis and Visualization of Genetic Variation,. EMBNEW.NEWS 4:14. Nishikata, M., Nishimori, I., Taniuchi, K., Takeuchi, T., Minakuchi, T., Kohsaki, T., Adachi, Y., Ohtsuki, Y., and Onishi, S. (2007). Carbonic anhydrase-related protein VIII promotes colon cancer cell growth. Mol Carcinog 46, 208-214. Nishimori, I., Takeuchi, T., Morimoto, K., Taniuchi, K., Okamoto, N., and Onishi, S. (2003). Expression of carbonic anhydrase-related protein VIII, X and XI in the enteric autonomic nervous system. Biomed Res 14, 70–74. Nishimori, I., Vullo, D., Minakuchi, T., Scozzafava, A., Capasso, C., and Supuran, C.T. (2013). Restoring catalytic activity to the human carbonic anhydrase (CA) related proteins VIII, X and XI affords isoforms with high catalytic efficiency and susceptibility to anion inhibition. Bioorg Med Chem Lett 23, 256-260. Nogradi, A., Jonsson, N., Walker, R., Caddy, K., Carter, N., and Kelly, C. (1997). Carbonic anhydrase II and carbonic anhydrase-related protein in the cerebellar cortex of normal and lurcher mice. Brain Res Dev Brain Res 98, 91-101. Ohradanova, A., Vullo, D., Kopacek, J., Temperini, C., Betakova, T., Pastorekova, S., Pastorek, J., and Supuran, C.T. (2007). Reconstitution of carbonic anhydrase activity of the cell-surface-binding protein of vaccinia virus. Biochem J 407, 61-67.

Okamoto, N., Fujikawa-Adachi, K., Nishimori, I., Taniuchi, K., and Onishi, S. (2001). cDNA sequence of human carbonic anhydrase-related protein, CA-RP X: mRNA expressions of CA-RP X and XI in human brain. Biochimica et biophysica acta 1518, 311-316. Ortutay C, Olatubosun A, Parkkila S, Vihinen M, Tolvanen M. (2010). An Evolutionary Analysis of Insect Carbonic Anhydrases. In Advances in Medicine and Biology Volume 7. Volume 7. Edited by Berhardt LV. New York: Nova Science Pub Inc;. 145-168. Paradis, E., Claude, J., and Strimmer, K. (2004). APE: Analyses of Phylogenetics and Evolution in R language. Bioinformatics 20, 289-290. Peles, E., Nativ, M., Campbell, P.L., Sakurai, T., Martinez, R., Lev, S., Clary, D.O., Schilling, J., Barnea, G., Plowman, G.D., et al. (1995). The carbonic anhydrase domain of receptor tyrosine phosphatase beta is a functional ligand for the axonal cell recognition molecule contactin. Cell 82, 251-260. Petersen, T.N., Brunak, S., von Heijne, G., and Nielsen, H. (2011). SignalP 4.0: discriminating signal peptides from transmembrane regions. Nat Methods 8, 785-786. Pfaffl, M.W. (2001). A new mathematical model for relative quantification in real-time RT- PCR. Nucleic Acids Res 29, e45. Picaud, S.S., Muniz, J.R., Kramm, A., Pilka, E.S., Kochan, G., Oppermann, U., and Yue, W.W. (2009). Crystal structure of human carbonic anhydrase-related protein VIII reveals the basis for catalytic silencing. Proteins 76, 507-511. Pierleoni, A., Martelli, P.L., and Casadio, R. (2008). PredGPI: a GPI-anchor predictor. BMC Bioinformatics 9, 392. Plassart, E., and Fontaine, B. (1994). Genes with triplet repeats: a new class of mutations causing neurological diseases. Biomed Pharmacother 48, 191-197. Pruitt, K.D., Tatusova, T., and Maglott, D.R. (2007). NCBI reference sequences (RefSeq): a curated non-redundant sequence database of genomes, transcripts and proteins. Nucleic Acids Res 35, D61-65. Puthussery, T., Gayet-Primo, J., and Taylor, W.R. (2011). Carbonic anhydrase-related protein VIII is expressed in rod bipolar cells and alters signaling at the rod bipolar to AII-amacrine cell synapse in the mammalian retina. Eur J Neurosci 34, 1419-1431. Ronquist, F., Teslenko, M., van der Mark, P., Ayres, D.L., Darling, A., Hohna, S., Larget, B., Liu, L., Suchard, M.A., and Huelsenbeck, J.P. (2012). MrBayes 3.2: efficient Bayesian phylogenetic inference and model choice across a large model space. Syst Biol 61, 539-542. Sievers, F., Wilm, A., Dineen, D., Gibson, T.J., Karplus, K., Li, W., Lopez, R., McWilliam, H., Remmert, M., Soding, J., et al. (2011). Fast, scalable generation of high-quality protein multiple sequence alignments using Clustal Omega. Mol Syst Biol 7, 539. Sjoblom, B., Elleby, B., Wallgren, K., Jonsson, B.H., and Lindskog, S. (1996). Two point mutations convert a catalytically inactive carbonic anhydrase-related protein (CARP) to an active enzyme. FEBS Lett 398, 322-325. Skaggs, L.A., Bergenhem, N.C., Venta, P.J., and Tashian, R.E. (1993). The deduced amino acid sequence of human carbonic anhydrase-related protein (CARP) is 98% identical to the mouse homologue. Gene 126, 291-292. Sly, W.S., and Hu, P.Y. (1995). Human carbonic anhydrases and carbonic anhydrase deficiencies. Annu Rev Biochem 64, 375-401. Spencer, W.C., Zeller, G., Watson, J.D., Henz, S.R., Watkins, K.L., McWhirter, R.D., Petersen, S., Sreedharan, V.T., Widmer, C., Jo, J., et al. (2011). A spatial and temporal map of C. elegans gene expression. Genome Res 21, 325-341. Supuran, C., Scozzafava A, and Conway, J, ed. (2004). Carbonic Anhydrase: Its Inhibitors and Activators (Washington, D.C.: CRC PR E S S).

68 Supuran, C.T. (2008). Carbonic anhydrases: novel therapeutic applications for inhibitors and activators. Nat Rev Drug Discov 7, 168-181. Suyama, M., Torrents, D., and Bork, P. (2006). PAL2NAL: robust conversion of protein sequence alignments into the corresponding codon alignments. Nucleic Acids Res 34, W609- 612. Tamura, K., Dudley, J., Nei, M., and Kumar, S. (2007). MEGA4: Molecular Evolutionary Genetics Analysis (MEGA) software version 4.0. Mol Biol Evol 24, 1596-1599. Taniuchi, K., Nishimori, I., Takeuchi, T., Fujikawa-Adachi, K., Ohtsuki, Y., and Onishi, S. (2002a). Developmental expression of carbonic anhydrase-related proteins VIII, X, and XI in the human brain. Neuroscience 112, 93-99. Taniuchi, K., Nishimori, I., Takeuchi, T., Ohtsuki, Y., and Onishi, S. (2002b). cDNA cloning and developmental expression of murine carbonic anhydrase-related proteins VIII, X, and XI. Brain Res Mol Brain Res 109, 207-215. Tashian, R.E. (1989). The carbonic anhydrases: widening perspectives on their evolution, expression and function. Bioessays 10, 186-192. Tashian, R.E.; Hewett-Emmett, D.; Carter, N.D. and Bergenhem, N.C.H. (2000). Carbonic anhydrase (CA)-related proteins (CA-RPs) and transmembrane proteins with CA or CA-RP domains. In: Chegwidden, W.R.; Carter, N.D. & Edwards, Y.H. (Eds.), Carbonic anhydrase (CA)-related proteins (CA-RPs) and transmembrane proteins with CA or CA-RP domains. The Carbonic Anhydases: New Horizons. Birkhauser, Basel, pp. 105–120. Tavernarakis, N., and Thireos, G. (1995). Transcriptional interference caused by GCN4 overexpression reveals multiple interactions mediating transcriptional activation. Mol Gen Genet 247, 571-578. Tolvanen, M.E., Ortutay, C., Barker, H.R., Aspatwar, A., Patrikainen, M., and Parkkila, S. (2013). Analysis of evolution of carbonic anhydrases IV and XV reveals a rich history of gene duplications and a new group of isozymes. Bioorg Med Chem 21, 1503-1510. Triezenberg, S.J., Kingsbury, R.C., and McKnight, S.L. (1988). Functional dissection of VP16, the trans-activator of herpes simplex virus immediate early gene expression. Genes Dev 2, 718-729. Turkmen, S., Guo, G., Garshasbi, M., Hoffmann, K., Alshalah, A.J., Mischung, C., Kuss, A., Humphrey, N., Mundlos, S., and Robinson, P.N. (2009). CA8 mutations cause a novel syndrome characterized by ataxia and mild mental retardation with predisposition to quadrupedal gait. PLoS Genet 5, e1000487. van de Leemput, J., Chandran, J., Knight, M.A., Holtzclaw, L.A., Scholz, S., Cookson, M.R., Houlden, H., Gwinn-Hardy, K., Fung, H.C., Lin, X., et al. (2007). Deletion at ITPR1 underlies ataxia in mice and spinocerebellar ataxia 15 in humans. PLoS Genet 3, e108. Wang, Z., & Moult, J. (2001). SNPs, protein structure, and disease. Hum Mutat 17, 263-270. Wen, F.C., Li, Y.H., Tsai, H.F., Lin, C.H., Li, C., Liu, C.S., Lii, C.K., Nukina, N., and Hsieh, M. (2003). Down-regulation of heat shock protein 27 in neuronal cells and non-neuronal cells expressing mutant ataxin-3. FEBS Lett 546, 307-314. Wu, C., Orozco, C., Boyer, J., Leglise, M., Goodale, J., Batalov, S., Hodge, C.L., Haase, J., Janes, J., Huss, J.W., 3rd, et al. (2009). BioGPS: an extensible and customizable portal for querying and organizing gene annotation resources. Genome Biol 10, R130. Yan, J., Jiao, Y., Jiao, F., Stuart, J., et al. (2007). Effects of carbonic anhydrase VIII deficiency on cerebellar gene expression profiles in the wdl mouse. Neurosci Lett 413, 196-201. Yoon , C.H. (1959). Waddler, a new mutation, and its interaction with quivering. J Hered 50, 238-244.

Original communications

70 Aspatwar et al. BMC Molecular Biology 2010, 11:25 http://www.biomedcentral.com/1471-2199/11/25

RESEARCH ARTICLE Open Access PhylogenyResearch article and expression of carbonic anhydrase-related proteins

Ashok Aspatwar*1,2,3, Martti EE Tolvanen1 and Seppo Parkkila2,3

Abstract Background: Carbonic anhydrases (CAs) are found in many organisms, in which they contribute to several important biological processes. The vertebrate α-CA family consists of 16 subfamilies, three of which (VIII, X and XI) consist of acatalytic proteins. These are named carbonic anhydrase related proteins (CARPs), and their inactivity is due to absence of one or more Zn-binding histidine residues. In this study, we analyzed and evaluated the distribution of genes encoding CARPs in different organisms using bioinformatic methods, and studied their expression in mouse tissues using immunohistochemistry and real-time quantitative PCR. Results: We collected 84 sequences, of which 22 came from novel or improved gene models which we created from genome data. The distribution of CARP VIII covers vertebrates and deuterostomes, and CARP X appears to be universal in the animal kingdom. CA10-like genes have had a separate history of duplications in the tetrapod and fish lineages. Our phylogenetic analysis showed that duplication of CA10 into CA11 has occurred only in tetrapods (found in mammals, frogs, and lizards), whereas an independent duplication of CA10 was found in fishes. We suggest the name CA10b for the second fish isoform. Immunohistochemical analysis showed a high expression level of CARP VIII in the mouse cerebellum, cerebrum, and also moderate expression in the lung, liver, salivary gland, and stomach. These results also demonstrated low expression in the colon, kidney, and Langerhans islets. CARP X was moderately expressed in the cerebral capillaries and the lung and very weakly in the stomach and heart. Positive signals for CARP XI were observed in the cerebellum, cerebrum, liver, stomach, small intestine, colon, kidney, and testis. In addition, the results of real-time quantitative PCR confirmed a wide distribution for the Car8 and Car11 mRNAs, whereas the expression of the Car10 mRNA was restricted to the frontal cortex, parietal cortex, cerebellum, midbrain, and eye. Conclusions: CARP sequences have been strongly conserved between different species, and all three CARPs show high expression in the mouse brain and CARP VIII is also expressed in several other tissues. These findings suggest an important functional role for these proteins in mammals.

Background vergent evolution of catalytic function. The animal king- Carbonic anhydrases (CAs), EC 4.2.1.1, are metal-con- dom CAs belong to a single gene family known as α-CAs taining enzymes that occur abundantly in nature and are that contain zinc as a metal ion in the active site. The found in almost all organisms that have been studied [1]. main chemical reaction catalyzed by CAs involves the - + CAs are fundamental to many biological processes, such reversible hydration of CO2 (CO2+H2O (HCO3 +H ). as photosynthesis, respiration, renal tubular acidification, In mammals, α-CAs are characterized by 16 different and bone resorption [2-5]. These enzymes are encoded isoforms, 13 of which (CA I, II, III, IV, VA, VB, VI, VII, by five distinct and evolutionarily unrelated gene families IX, XII, XIII, XIV, and XV) are enzymatically active, known as α, β, γ, δ, and ζ CAs [6]. Interestingly, there is whereas the other 3, namely, the CA-related proteins no sequence similarity between these different families. (CARPs) VIII, X, and XI, appear to lack CA activity Thus, the CA families are excellent examples of the con- because of substitutions to 1 or more of the 3 functionally important histidine residues [3,7,8]. In addition, the * Correspondence: [email protected] receptor-type protein tyrosine phosphatases β and γ 1 Bioinformatics Group, Institute of Medical Technology, 33014 University of Tampere, Tampere, Finland (RPTP β, RPTP γ) also contain 'CA-like' domains [3,9]. Full list of author information is available at the end of the article The 13 active CA isozymes differ in their subcellular

© 2010 Aspatwar et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons BioMed Central Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Aspatwar et al. BMC Molecular Biology 2010, 11:25 Page 2 of 19 http://www.biomedcentral.com/1471-2199/11/25

localizations such that CAs I, II, III, VII, and XIII are all vertebrates, and studied the distribution of the three cytosolic enzymes, CAs IV, IX, XII, XIV, and XV are all CARP mRNAs using real-time quantitative PCR (qPCR) membrane-associated enzymes, CAs VA and VB are and the corresponding proteins using immunohis- mitochondrial, and CA VI is a secreted protein. tochemistry in different mouse tissues. Previous studies on the distribution of CARPs using either western blot analysis or RT-PCR methods have Results shown a wide expression profile in all parts of the brain in Bioinformatic survey and comparison of CARP sequences both humans and mice [10-13]. Notably, immunohis- Detailed information and database sources from which tochemical studies on CARPs have been mainly focused sequences were obtained are shown in Table 1. In total, on brain tissues. The results have shown predominant 84 full-length sequences were obtained from 38 organ- expression of CARP VIII in the mouse and human cere- isms (3 invertebrates and 35 vertebrates). Thirty-three bellum, especially in the Purkinje cells. Studies on CARP gene sequences encoding for CARP VIII were identified, X and XI have revealed a lower level of expression in the of which 3 sequences were from deuterostome inverte- cerebellum [14-16]. Previous investigations using reverse brates, namely, Branchiostoma floridae, Trichoplax transcription polymerase chain reaction (RT-PCR), adhaerens, and Strongylocentrotus purpuratus, and 30 northern blot analysis, western blot assays or dot blots were from vertebrates. We identified 31 sequences of have reported restricted expression of all CARPs in some CARP X and 19 sequences of CARP XI in these verte- mouse and human tissues including the brain [13,17]. brates, which included a single CARP X gene from a The presence of CARPs in the human and mouse brain chordate (Branchiostoma floridae). CARP X-like has suggested important roles for these proteins in the sequences in invertebrates were discovered in both pro- brain development and/or neural functions [12,16]. Inter- tostomes and deuterostomes. A detailed analysis of inver- estingly, CARP VIII and XI may also be involved in can- tebrate CARP X homologs will be reported elsewhere. cer development in the gastrointestinal tract and lungs Multiple sequence alignments (MSAs) of the CARP VIII, [13,18-21]. X, and XI protein sequences are presented in Figures 1, 2, The pivotal physiological function of CARP VIII and 3, respectively. became clearly evident in recently published reports on Among vertebrate sequences, the protein sequence CARP VIII mutations in both human and mouse. A study identities ranged from 67% to 100% for CARP VIII, from of waddle mice characterized by a spontaneous mutation 70% to 100% for CARP X and from 70% to 100% for in the Car8 gene showed ataxia and a distinctive lifelong CARP XI. The 100% values were observed between pri- gait disorder [22]. Another recent study described mild mate sequences. However, even between humans and mental retardation, quadrupedal gait and ataxia in mem- mice, identities of CARP orthologs were 98%, 100%, and bers of an Iraqi family who each possessed a defect in the 96% for CARP VIII, X, and XI, respectively. When the CA8 gene [23]. These studies clearly indicated that CARP invertebrate CARP VIII and CARP X sequences used in VIII plays an important role in motor coordination. this study were compared to their vertebrate orthologs, The three-dimensional structure of CARP VIII has the sequence identities were only 40% to 45%. For com- been recently solved [24], as the only protein in the CARP parison, in case of enzymatically active CAs, protein subfamily. The structural basis of catalytic inactivity is sequence identity between human and mouse are 94%, confirmed in this study, but currently there is no inter- 93%, 92%, and 91% for CAs VII, Vb, III, and XIII, respec- pretation to correlate the structure to any function. tively, and 50% to 83% for other pairs of isozymes. Thus, The CARP sequences are well conserved throughout all sequence conservation was found to be higher in all vertebrates, suggesting that CARPs may play biologically CARPs than in any of the active CAs. important roles in higher organisms. The number of members in the CARP families has increased with the Phylogenetic analysis completion of vertebrate genome sequencing projects, The phylogenetic tree of the CARP protein sequences is but many sequences have been annotated in incomplete shown in Figure 4. The full MSA from which the tree was or even partly incorrect forms. To date there are no sys- calculated is provided as Additional file 1. The tree shows tematic studies on the distribution and phylogenetic anal- two distinct sequence pools, including one pool for ysis of CARP genes across species nor does there exist CARP VIII and the other pool for CARPs X and XI. The any parallel study on the distribution of all three CARP CARP VIII pool subscribes to the expected animal taxon- proteins and their mRNA levels in mouse tissues. There- omy, except for mammals, which are not resolved due to fore, we first used bioinformatic methods to identify and sequence identities near 100%. The second sequence pool evaluate the distribution of CARP genes across different is comprised of the CARP X and CARP XI sequences. species. We subsequently performed sequence and phy- CARP X forms three subgroups, including the large sub- logenetic analysis of CARPs VIII, X, and XI, focusing on group consisting of sequences from mammals, frogs, and Aspatwar et al. BMC Molecular Biology 2010, 11:25 Page 3 of 19 http://www.biomedcentral.com/1471-2199/11/25

Table 1: Details of the carbonic anhydrase-related protein (CARP) sequences used in this study

Group Organism Scientific Name CARPs Acc. No./ID Length (aa) Description1

Cow Bos taurus VIII NP_001077159 290 NCBI_RefSeq XA0JN41 328Uniprot XI NP_783648.1 328 NCBI_RefSeq Marmoset Callithrix jacchus VIII - 290 UCSC_BLAT X- 328UCSC_BLAT XI A6MLD1 321 Uniprot _UCSC_BLAT Dog Canis familiaris VIII XP_544094.2 290 NCBI_RefSeq X XP_866307.1 328 Ensembl_UCSC_BLAT XI XP_852031.1 328 NCBI_RefSeq Guinea Pig Cavia porcellus VIII ENSCPOP00000017966 256 Ensembl X ENSCPOP00000013970 328 Ensembl XI ENSCPOP00000011461 332 Ensembl Armadillo Dasypus novemcinctus X ENSDNOP00000008341 327 Ensembl Horse Equus caballus VIII XP_001496523.1 290 NCBI_RefSeq X XP_001503286.2 328 NCBI_RefSeq XI ENSECAP00000015876 278 Ensembl Cat Felis catus X ENSFCAP00000002102 326 Ensembl_UCSC_BLAT XI ENSFCAP00000006798 265 Ensembl Human Homo sapiens VIII P35219 290 Uniprot XQ9NS85328Uniprot XI O75493 328 Uniprot Mouse Mus musculus VIII P28651 291 Uniprot X P61215 328 Uniprot XI O70354 328 Uniprot Macaque Macaca mulatta VIII XP_001088977.1 290 NCBI_RefSeq X XP_001101492.1 334 NCBI_RefSeq XI XP_001113730.1 328 RefSeq_UCSC_BLAT Rhesus Monkey Macaca fascicularis X Q9N085 328 Uniprot Mouse Lemur Microcebus murinus VIII ENSMICP00000001089 286 Ensembl Mammals Opossum Monodelphis domestica VIII ENSMODP00000010658 289 Ensembl_UCSC_BLAT X ENSMODP00000015705 328 Ensembl_UCSC_BLAT XI ENSMODP00000017625 332 Ensembl Microbat Myotis lucifugus VIII ENSMLUP00000008230 275 Ensembl Pika Ochotonidae princeps VIII ENSOPRP00000014387 290 Ensembl Platypus Ornithorhynchus VIII ENSOANP00000008002 267 Ensembl anatinus X ENSOANP00000003611 308 Ensembl_UCSC_BLAT Rabbit Oryctolagus cuniculus X ENSOCUP00000002025 333 Ensembl Bushbaby Otolemur garnettii X ENSOGAP00000004458 327 Ensembl Chimpanzee Pan troglodytes VIII XP_519778.2 290 NCBI_RefSeq X XP_001171455.1 328 NCBI_RefSeq XI XP_001171520.1 328 RefSeq_UCSC_BLAT Sumatran Pongo abelii VIII - 290 UCSC_BLAT Orangutan X Q5R4U0 328 Uniprot XI NP_001128968.1 328 NCBI_RefSeq Aspatwar et al. BMC Molecular Biology 2010, 11:25 Page 4 of 19 http://www.biomedcentral.com/1471-2199/11/25

Table 1: Details of the carbonic anhydrase-related protein (CARP) sequences used in this study (Continued)

Orangutan Pongo pygmaeus VIII ENSPPYP00000020875 290 Ensembl X ENSPPYP00000009287 328 Ensembl XI ENSPPYP00000011415 328 Ensembl Rat Rattus norvegicus VIII Q5PPN4 290 Uniprot X EDM05681 319 NCBI_RefSeq XI NP_783639 328 NCBI_RefSeq Pig Sus scrofa VIII XP_001926916 291 NCBI_RefSeq_EST XI Q866X6 331 Uniprot Tarsier Tarsius syrichta VIII ENSTSYP00000009029 256 Ensembl Dolphin Tursiops truncatus VIII ENSTTRP00000015250 256 Ensembl X ENSTTRP00000001587 308 Ensembl XI ENSTTRP00000007758 320 Ensembl Sheep Ovis aries XI Q95203 328 Uniprot

Anole Lizard Anolis carolinensis VIII - 257 UCSC_BLAT X- 321UCSC_BLAT XI ENSACAP00000015571 289 Ensembl Chicken Gallus gallus VIII ENSGALP00000024873 283 Ensembl X XP_415644.1 328 NCBI_RefSeq Zebra Finch Taeniopygia guttata VIII ENSTGUP00000011522 258 Ensembl X ENSTGUP00000009733 328 Ensembl Frog Xenopus tropicalis VIII Q5M8Z5 282 Uniprot X ENSXETP00000002773 321 Ensembl_UCSC_BLAT XI - 303 UCSC_BLAT Zebrafish Danio rerio VIII NP_001017571.1 281 NCBI_RefSeq Other Xa NP_001032198.1 328 NCBI_RefSeq vertebrates Xb XP_696967.2 308 NCBI_RefSeq Stickleback Gasterosteus aculeatus VIII ENSGACP00000019684 281 Ensembl Xa ENSGACP00000020102 323 Ensembl_UCSC_BLAT Xb ENSGACP00000000619 328 Ensembl Rainbow trout Oncorhynchus mykiss VIII Q5I2J1 281 Uniprot Medaka Oryzias latipes VIII ENSORLP00000016815 281 Ensembl Fugu Takifugu rubripes VIII ENSTRUP00000009702 282 Ensembl Xa ENSTRUP00000007839 297 Ensembl_UCSC_BLAT Xb ENSTRUP00000032938 328 Ensembl_UCSC_BLAT Pufferfish Tetraodon nigroviridis VIII ENSTNIP00000005959 272 Ensembl Xa ENSTNIP00000017020 328 Ensembl_UCSC_BLAT

Lancelet Branchiostoma lancelet VIII C3XTT7 256 Uniprot_UCSC_BLAT X- 319UCSC_BLAT Invertebrates Trichoplax Trichoplax adhaerens VIII XP_002111595.1 348 NCBI_RefSeq Sea urchin Strongylocentrotus VIII XP_795365.2 312 NCBI_RefSeq purpuratus 1 The sources of the best protein sequences. NCBI, RefSeq, UniProt and Ensembl refer to the respective databases, and UCSC_BLAT refers to discovery of novel genes/exons using the UCSC genome browser and subsequent gene model building, as detailed in Materials and Methods. EST means that a further EST sequence was used to extend an incomplete genome sequence. Aspatwar et al. BMC Molecular Biology 2010, 11:25 Page 5 of 19 http://www.biomedcentral.com/1471-2199/11/25

* 20 * 40 * 60 * 80 * 100 CAH8_Drer : ---MADNVIE------ESGNY-PGKDELDWGYEE------G-VEWGLLFPEANGEYQSPINLNSREARYDPQLLDVGLNPNYVVCRDCEVIND : 76 CAH8_Omyk : ---MADSVIE------ESDYY-PGKDELDWGYQG------G-VEWGLLFPDANGEYQSPINLNSREARYDPLLLDEGLTPNYVVCRDCEVIND : 76 CAH8_Gacu : ---MADSVNE------ESDYN-PGKDELAWGYEE------G-VEWGLHFPSANGEYQSPINLNSREAEYDPSLLDAGLSPNYVVCRECEVIND : 76 CAH8_Olat : -LCMGGGVEESLSFLQRRQNDQR-LRKDELDWGYEE------G-VEWGLHFPAANGEYQSPINLNSREAQYDPSLLDVGLSPNYVVCRDCEVVND : 86 CAH8_Trub : ---MADSINE------ELDYKESGNEELDWGYEE------G-VEWGLLFPSANGEYQSPINLNSREAQYDPSLLDVGLSPNYVVCRDCEVIND : 77 CAH8_Tnig : --LVQES------WYLKGVSPLS------G-VEWGLLFPSANGEYQSPINLNSREARYDPALLDVGLAPNYLVCRDCEVLND : 67 CAH8_Ggal : --MADRSLIEGAEPFPRREEAP------EWGYEE------G-VEWGLIFPDANGEYQSPINLNSREAKYDPSLLEVRLSPNYVVCRDCEVIND : 78 CAH8_Tgut : ------S------G-VEWGLIFPDANGEYQSPINLNSREAKYDPSLLEVRLSPNYVVCRDCEVIND : 53 CAH8_Cpor : ------GVEWGLVFPDANGEYQSPINLNSREARYDPSLLDVRLSPNYVVCRDCEVTND : 52 CAH8_Cfam : --MADLSFIEDSVAFRGYEEGGVLNRK-LHQWGWPE------GGVEWGLVFPDANGEYQSPINLNSREARYDPSLLDVRLSPNYVVCRDCEVTND : 86 CAH8_Rnor : --MADLSFIEDAVAFPEKEEDEEEEEE-GVEWGYEE------G-VEWGLVFPDANGEYQSPINLNSREARYDPSLLDVRLSPNYVVCRDCEVTND : 85 CAH8_Mmus : --MADLSFIEDAVAFPEKEEDEEEEEEEGVEWGYEE------G-VEWGLVFPDANGEYQSPINLNSREARYDPSLLDVRLSPNYVVCRDCEVTND : 86 CAH8_Btau : --MADLSFIEDSVAFPEKEEDEEEEEE-GVEWGYEE------G-VEWGLVFPDANGEYQSPINLNSREARYDPSLLDIRLSPNYVVCRDCEVAND : 85 CAH8_Mmur : -----LSFIEDSVAF-EKEEDEEEEEE-GVEWGYEE------G-VEWGLVFPDANGEYQSPINLNSREARYDPSLLDIRLSPNYVVCRDCEVTND : 81 CAH8_Opri : --MADLSFIEDSVAFPEKEEDEEEEEE-GVEWGYEE------G-VEWGLVFPDANGEYQSPINLNSREARYDPSLLDVRLSPNYVVCRDCEVTND : 85 CAH8_Ecab : --MADLSFIEDSVAFPEKEEDEEEEEE-GVEWGYEE------G-VEWGLVFPDANGEYQSPINLNSREARYDPSLLDVRLSPNYVVCRDCEVTND : 85 CAH8_Sscr : --MADLSFIEDSVAFPEKEEDEEEEEEEGVEWGYEE------G-VEWGLVFPDANGEYQSPINLNSREARYDPSLLDVRLSPNYVVCRDCEVTND : 86 CAH8_Pabe : --MADLSFIEDTVAFPEKEEDEEEEEE-GVEWGYEE------G-VEWGLVFPDANGEYQSPINLNSREARYDPSLLDVRLSPNYVVCRDCEVTND : 85 CAH8_Ptro : --MADLSFIEDTVAFPEKEEDEEEEEE-GVEWGYEE------G-VEWGLVFPDANGEYQSPINLNSREARYDPSLLDVRLSPNYVVCRDCEVTND : 85 CAH8_Mmul : --MADLSFIEDTVAFPEKEEDEEEEEE-GVEWGYEE------G-VEWGLVFPDANGEYQSPINLNSREARYDPSLLDVRLSPNYVVCRDCEVTND : 85 CAH8_Hsap : --MADLSFIEDTVAFPEKEEDEEEEEE-GVEWGYEE------G-VEWGLVFPDANGEYQSPINLNSREARYDPSLLDVRLSPNYVVCRDCEVTND : 85 CAH8_Cjac : --MADLSFIEDTVAFPEKEDDEEEEEE-GVEWGYEE------G-VEWGLVFPDANGEYQSPINLNSREARYDPSLLDVRLSPNYVVCRDCEVTND : 85 CAH8_Ppyg : --MADLSFIEDTVAFPEKEEDEEEEDE-GVEWGYEE------G-VEWGLVFPDANGEYQSPINLNSREARYDPSLLDVRLSPNYVVCRDCEVTND : 85 CAH8_Tsyr : ------VEWGLVFPDANGEYQSPINLNSREARYDPSLLDVRLSPNYVVCRDCEVTND : 51 CAH8_Ttru : ------VEWGLVFPDANGEYQSPINLNSREARYDPSLLDVRLSPNYVVCRDCEVAND : 51 CAH8_Mluc : ------TFPQENEQGGEGVE-----STEE------G-VEWGLVFPDANGEYQSPINLNSREARYDPSLLDVRLSPNYVVCRDCEVTND : 70 CAH8_Mdom : ----SLTFCKTNLSVSELKSDSQPGEK---KWGYEE------G-VEWGLVFPDANGEYQSPINLNSREARYDPSLLDIRLSPNYVVCRDCEVTND : 81 CAH8_Oana : -----LSWGFETLFL------G-VEWGLVFPDANGEYQSPINLNSREARYDPSLLEVRLSPNYVVCRDCEVTND : 62 CAH8_Acar : ------G-VEWGLIFPDANGEYQSPINLNSREAKYDPLLLEVSLSPNYVVCRDCEVIND : 52 CAH8_Xtro : --MADKHYIED------EAVQGEKENQEWGYEE------G-VEWGLLYPEANGDYQSPININSREAMYDPSLLEVRLTPSYVVCRDCEVIND : 77 CAH8_Spur : MSILKFTKFRKRYLRGKLLTRRSDSREKLIPWVHEDTFDDLLCSLPIKAFSYRGTAEWGIHFQEANGDCQSPIDINSREAVYREGLNQPPLEVNYALCRDCDLINT : 106 CAH8_Blan : ------EWGLHWPYANGDQQSPVNINSRTATFDETLAERPLLISYCLCREWELSND : 50 CAH8_Tadh : ------MILGPSRWNDYFPAAKGNRQSPININVDNQHCDGDLKEDPLDIRYIHSRTNAIKNT : 56 * 120 * 140 * 160 * 180 * 200 * CAH8_Drer : GHTVRIMLKSKSVVTGGPLPSDHEYELSEVRFHWGKENQRGSEHTVNFKAFPMELHLIHWNSTLFNSVEEAMGKKRGILIIALFVQVGKEHLGLKAITDVLQDLQY : 182 CAH8_Omyk : GHTVRIMLRSKSVVTGGPLQIDHEYELHEVRFHWGKENQRGSEHTVNFKAFPMELHLIHWNTTLFSSVEEALGKTNGVLIIALFVQIGKEHLGLKAITEVLQDLQY : 182 CAH8_Gacu : GHTVRIILKSKSVVSGGPLPSDHEYELHEVRFHWGKENQRGSEHTVNFKAFPMELHLIHWNSTLFNTLEDALGKKNGVLTIALFVQIGKEHLGLKAVTEVLQDLQY : 182 CAH8_Olat : GHTVRILLKSKSVVTGGPLPSDHEYELHEVRFHWGKENQRGSEHTVNFKAFPMELHLIHWNSTLFNSLEDALGRKNGVLIIALFVQVGKEHLGLKAITEVLQDLQY : 192 CAH8_Trub : GHTVRIILKSKSVVTGGPLPSGHEYELHEVRFHWGKENQRGSEHTVNFKAFPMELHLIHWNSTLFNTLEDALGKKKKSILAQEFVQIGKEHLGLKAITEVLQDLQY : 183 CAH8_Tnig : GHTVRILLRSKSVVAGGPLPSGHEYELHEVRFHWGKENQRGSEHTVNFKAFPMELHLIHWNTTLFSTLEDALGQKNGVLIIALFVQIGKEHLGLKAITEVLQDLQY : 173 CAH8_Ggal : GHSIQIALKSKSVLIGGPLPRGHEFELHDVRFHWGRENQRGSEHTVNFKAFPMELHLMHWNSTLYSSIDEAVGKKHGIAIIALFVQIGKEHVGLKAVTEILQDIQY : 184 CAH8_Tgut : GHSIQIALKSKSVLIGGPLPRGHEFELHDVRFHWGRENQRGSEHTVNFKAFPMELHLMHWNSTLYSSIDEAVGKKHGIAIIALFVQIGKEHVGLKAVTEILQDIQY : 159 CAH8_Cpor : GHTIQVILKSKSVLSGGPLPQGHEFELYEVRFHWGRENQRGSEHTVNFKAFPMELHLIHWNSTLFGSVDEAVG-PHGIAIIALFVQIGKEHVGLKAVTEILQDIQY : 157 CAH8_Cfam : GHTIQVILKSKSVLSGGPLPQGHEFELYEVRFHWGRENQRGSEHTVNFKAFPMELHLIHWNSTLFGSIDEAVGKPHGIAIIALFVQIGKEHVGLKAVTEILQDIQY : 192 CAH8_Rnor : GHTIQVILKSKSVLSGGPLPQGQEFELYEVRFHWGRENQRGSEHTVNFKAFPMELHLIHWNSTLFGSIDEAVGKPHGIVIIALFVQIGKEHVGLKAVTEILQDIQY : 191 CAH8_Mmus : GHTIQVILKSKSVLSGGPLPQGQEFELYEVRFHWGRENQRGSEHTVNFKAFPMELHLIHWNSTLFGSIDEAVGKPHGIAIIALFVQIGKEHVGLKAVTEILQDIQY : 192 CAH8_Btau : GHTIQVILKSKSVLSGGPLPQGHEFELYEVRFHWGRENQRGSEHTVNFKAFPMELHLIHWNSTLFGSIDEAVGKPHGIAIIALFVQIGKEHVGLKAVTEILQDIQY : 191 CAH8_Mmur : GHTIQVILKSKSVLSGGPLPQGHEFELYEVRFHWGRENQRGSEHTVNFKAFPMELHLIHWNSTLFGSIDEAVGKPHGIAIIALFVQIGKEHVGLKAVTEILQDIQY : 187 CAH8_Opri : GHTIQVILKSKSVLSGGPLPQGHEFELYEVRFHWGRENQRGSEHTVNFKAFPMELHLIHWNSTLFGSIDEAVGKPHGIAIIALFVQIGKEHVGLKAVTEILQDIQY : 191 CAH8_Ecab : GHTIQVILKSKSVLLGGPLPQGHEFELYEVRFHWGRENQRGSEHTVNFKAFPMELHLIHWNSTLFSSIDEAVGKPHGIAIIALFVQIGKEHAGLKAVTEILQDIQY : 191 CAH8_Sscr : GHTIQVILKSKSVLSGGPLPQGHEFELYEVRFHWGRENQRGSEHTVNFKAFPMELHLIHWNSTLFGSIDEAVGKPHGIAIIALFVQIGKEHVGLKAVTEILQDIQY : 192 CAH8_Pabe : GHTIQVILKSKSVLSGGPLPQGHEFELYEVRFHWGRENQRGSEHTVNFKAFPMELHLIHWNSTLFGSIDEAVGKPHGIAIIALFVQIGKEHVGLKAVTEILQDIQY : 191 CAH8_Ptro : GHTIQVILKSKSVLSGGPLPQGHEFELYEVRFHWGRENQRGSEHTVNFKAFPMELHLIHWNSTLFGSIDEAVGKPHGIAIIALFVQIGKEHVGLKAVTEILQDIQY : 191 CAH8_Mmul : GHTIQVILKSKSVLSGGPLPQGHEFELYEVRFHWGRENQRGSEHTVNFKAFPMELHLIHWNSTLFGSIDEAVGKPHGIAIIALFVQIGKEHVGLKAVTEILQDIQY : 191 CAH8_Hsap : GHTIQVILKSKSVLSGGPLPQGHEFELYEVRFHWGRENQRGSEHTVNFKAFPMELHLIHWNSTLFGSIDEAVGKPHGIAIIALFVQIGKEHVGLKAVTEILQDIQY : 191 CAH8_Cjac : GHTIQVILKSKSVLSGGPLPQGHEFELYEVRFHWGRENQRGSEHTVNFKAFPMELHLIHWNSTLFGSIDEAVGKPHGIAIIALFVQIGKEHVGLKAVTEILQDIQY : 191 CAH8_Ppyg : GHTIQVILKSKSVLSGGPLPQGHEFELYEVRFHWGRENQRGSEHTVNFKAFPMELHLIHWNSTLFGSIDEAVGKPHGIAIIALFVQIGKEHVGLKAVTEILQDIQY : 191 CAH8_Tsyr : GHTIQVILKSKSVLSGGPLPQGHEFELYEVRFHWGRENQRGSEHTVNFKAFPMELHLIHWNSTLFGSIDEAVGKPHGIAIIALFVQIGKEHVGLKAVTEILQDIQY : 157 CAH8_Ttru : GHAIQVILKSKSVLSGGPLPQGHEFELYEVRFHWGRENQRGSEHTVNFKAFPMELHLIHWNSTLFGSIDEAVGKPRGIAIIALFVQIGKEHVGLKAVTEILQDIQY : 157 CAH8_Mluc : GHTIQVILKSKSVLSGGPLPQGHEFELYEVRFHWGRENQRGSEHTVNFKAFPMELHLIHWNSTLFGSIDEAVGKPHGIAIIALFVQIGKEHVGLKAVTEILQDIQY : 176 CAH8_Mdom : GHTIQIVLKSKSVLTGGPLPQGHEFELYEVRFHWGRENQRGSEHTVNFKAFPMELHLIHWNSTLYSSIDEAVGKTHGIAIIALFVQIGKEHIGLKAVTDVLQDIQY : 187 CAH8_Oana : GHTIQIIFKSKSVLAGGPLPRGHEFELHEVRFHWGRENQRGSEHTVNFKAFPMELHLIHWNSTLYSSIDEAVGKTHGVAIIALFVQIGKEHVGLKAVTDILQDIQY : 168 CAH8_Acar : GHSVQILLKSKSVLVGGPLPRGHEFELHDVRFHWGRENQRGSEHTVNFKAFPMELHLVHWNSTMYHNIDEAIGKKNGIAIIALFVQIGKEHSGLKTVTEILQDIQY : 158 CAH8_Xtro : GHVVQILLKSKSVLKGGPLPRGHEYELNEVRFHWGKENQRGSEHTVNFKAFPMELHLIHWNSTLYRSLEEAMGKVHGIVIISLFVQIGKENIGLKAITEVLQDIFY : 183 CAH8_Spur : GTGIQIIFKYKPVLSGGPLPQNSKFELAACAFHWGQKDDRGSEHTVNFKAYPMELHLIHWNSSTYSTLEEAMGKPHGISIIALFIQVGREHAGLRTFTDYLEAVQY : 212 CAH8_Blan : GHAVQVVPKPKTVTSGGPLPEGHEFELCHLRFHWGREDDRGSEHTVNFKAFPMELHLLHWNTSLYSNLADAMGKRDGIVIMSVIVQIGRENAGVKNFTDHLEDIQY : 156 CAH8_Tadh : GNTIRISFSPGSTIQGGPL--EHQYELLHCSFHWGKTNSRGSEHTVIGKQFPMEVQLLHWNTSLYKQAEDAMTAPQGLAIMAAFIQIGQENPDLKAITDVITEVRY : 160 220 * 240 * 260 * 280 * 300 * CAH8_Drer : KGKTKIIPC-FNPNTLLP---DPLLRDYWVYEGSLTTPPCSENVTWILYRYPLTISQMQIEEFRRLRPHVKGAEL-PEGNDGMLGDNFRPTQPLSDRVVRAAFQ : 281 CAH8_Omyk : KGKTKIIPC-FNPNTLLP---DPLLRDYWVYEGSLTTPPCSESVTWILYRYPLTISQLQIEEFRRLRSHLKGADL-PEGNDGMLGDNFRPSQPLSDRTVRAAFQ : 281 CAH8_Gacu : KGKNKIIPC-FNPNTLLP---DPLLRDYWVYEGSLTTPPCSERVTWILYRYPLTISQSQIEEFRRLRSHVKGAEL-LEGNDGMLGDNFRPTQPLSDRTVRAAFQ : 281 CAH8_Olat : KGKSKIIPC-FNPNTLLP---DPLLRDYWVYDGSLTTPPCSENVTWILYRYPLTISQMQIEEFRRLRSHVKGAEL-LEGNDGMLGDNFRPTQPLSDRTVRAAFQ : 291 CAH8_Trub : KGKNKLIPC-FNPNTLLP---DPLLRDYWVYEGSLTTPPCSEKVTWILFRYPLTISQIQIEEFRRLRSHVKGAEL-LEGNDGLLGDNFRPTQPLSDRTVRAAFQ : 282 CAH8_Tnig : KGKSKIIPC-FNPNTLLP---DPLLRDYWVYEGSLTTPPCSEKVTWILFRYPLTISQVQIEEFRRLRSHIKGAEL-LEGNDGLLGDNFRPTQPLSDRMVRAAFQ : 272 CAH8_Ggal : KGKSKTIPC-FNPNSLLP---DPLLRDYWVYEGSLTIPPCSEGVTWILFRYPLTVSQVQIEEFRRLRTHVKGAEL-LEGCDGILGDNFRPTQPLSDRVIRAAFQ : 283 CAH8_Tgut : KGKSKTIPC-FNPNSLLP---DPLLRDYWVYEGSLTIPPCSEGVTWILFRYPLTVSQVQIEEFRRLRTHVKGAEL-LEGSDGILGDNFRPTQPLSDRVIRAAFQ : 258 CAH8_Cpor : KGKSKTIPC-FNPNTLLP---DPLLRDYWVYDGSLTIPPCSEGVTWILFRYPLTISQLQIEEFRRLRTHVKGAEL-VEGCDGILGDNFRPTQPLSDRVIRAAFQ : 256 CAH8_Cfam : KGKSKTIPC-FNPNTLLP---DPLLRDYWVYEGSLTIPPCSEGVTWILFRYPLTISQLQIEEFRRLRTHVKGAEL-VEGCDGILGDNFRPTQPLSDRVIRAAFQ : 291 CAH8_Rnor : KGKSKTIPC-FNPNTLLP---DPLLRDYWVYEGSLTIPPCSEGVTWILFRYPLTISQLQIEEFRRLRTHVKGAEL-VEGCDGILGDNFRPTQPLSDRVIRAFSS : 290 CAH8_Mmus : KGKSKTIPC-FNPNTLLP---DPLLRDYWVYEGSLTIPPCSEGVTWILFRYPLTISQMQIEEFRRLRTHVKGAEL-VEGCDGILGDNFRPTQPLSDRVIRAAFQ : 291 CAH8_Btau : KGKSKTIPC-FNPNTLLP---DPLLRDYWVYEGSLTIPPCSEGVTWILFRYPLTISQLQIEEFRRLRTHVKGAEL-VEGCDGILGDNFRPTQPLSDRVIRAAFQ : 290 CAH8_Mmur : KGKSKTIPC-FNPNTLLP---DPLLRDYWVYEGSLTIPPCSEGVTWILFRYPLTISQLQIEEFRRLRTHVKGAEL-VEGCDGILGDNFRPTQPLSDRVIRAAFQ : 286 CAH8_Opri : KGKSKTIPC-FNPNTLLP---DPLLRDYWVYEGSLTIPPCSEGVTWILFRYPLTISQLQIEEFRRLRTHVKGAEL-VEGCDGILGDNFRPTQPLSDRVIRAAFQ : 290 CAH8_Ecab : KGKSKTIPC-FNPNTLLP---DPLLRDYWVYEGSLTIPPCSEGVTWILFRYPLTISQLQIEEFRRLRTHVKGAEL-VEGCDGILGDNFRPTQPLSDRVIRAAFQ : 290 CAH8_Sscr : KGKSKTIPC-FNPNTLLP---DPLLRDYWVYEGSLTIPPCSEGVTWILFRYPLTISQLQIEEFRRLRTHVKGAEL-VEGCDGILGDNFRPTQPLSDRVIRAAFQ : 291 CAH8_Pabe : KGKSKTIPC-FNPNTLLP---DPLLRDYWVYEGSLTIPPCSEGVTWILFRYPLTISQLQIEEFRRLRTHVKGAEL-VEGCDGILGDNFRPTQPLSDRVIRAAFQ : 290 CAH8_Ptro : KGKSKTIPC-FNPNTLLP---DPLLRDYWVYEGSLTIPPCSEGVTWILFRYPLTISQLQIEEFRRLRTHVKGAEL-VEGCDGILGDNFRPTQPLSDRVIRAAFQ : 290 CAH8_Mmul : KGKSKTIPC-FNPNTLLP---DPLLRDYWVYEGSLTIPPCSEGVTWILFRYPLTISQLQIEEFRRLRTHVKGAEL-VEGCDGILGDNFRPTQPLSDRVIRAAFQ : 290 CAH8_Hsap : KGKSKTIPC-FNPNTLLP---DPLLRDYWVYEGSLTIPPCSEGVTWILFRYPLTISQLQIEEFRRLRTHVKGAEL-VEGCDGILGDNFRPTQPLSDRVIRAAFQ : 290 CAH8_Cjac : KGKSKTIPC-FNPNTLLP---DPLLRDYWVYEGSLTIPPCSEGVTWILFRYPLTISQLQIEEFRRLRTHVKGAEL-VEGCDGILGDNFRPTQPLSDRVIRAAFQ : 290 CAH8_Ppyg : KGKSKTIPC-FNPNTLLP---DPLLRDYWVYEGSLTIPPCSEGVTWILFRYPLTISQLQIEEFRRLRTHVKGAEL-VEGCDGILGDNFRPTQPLSDRVIRAAFQ : 290 CAH8_Tsyr : KGKSKTIPC-FNPNTLLP---DPLLRDYWVYEGSLTIPPCSEGVTWILFRYPLTISQLQIEEFRRLRTHVKGAEL-VEGCDGILGDNFRPTQPLSDRVIRAAFQ : 256 CAH8_Ttru : KGKSKTIPC-FNPNTLLP---DPLLRDYWVYEGSLTIPPCSEGVTWILFRYPLTISQLQIEEFRRLRTHAKGAEL-VEGCDGILGDNFRPTQPLSDRVIRAAFQ : 256 CAH8_Mluc : KGKSKIIPC-FNPNTLLP---DPLLRDYWVYEGSLTIPPCSEGVTWILFRYPLTISQLQIEEFRRLRTHVKGAEL-VEGCDGILGDNFRPTQPLSDRVIRAAFQ : 275 CAH8_Mdom : KGKTKIIPC-FNPNTLLPEKPDPLLRDYWVYEGSLTIPPCSEGVTWILFRYPLTISQIQIEEFRRLRTHVKGAEL-LEGCDGILGDNFRPTQPLSDRVIRAAFQ : 289 CAH8_Oana : KGKSKTIPC-FNPNTLLP---DPLLRDYWVYEGSLTIPPCSEGVTWILFRYPLTISQIQIEEFRRLRTHIKGAEL-VEGCDGILGDNFRPTQPLSDRVIRAAFQ : 267 CAH8_Acar : KGKSKTIPC-FNPNSLLP---DPLLRDYWVYEGSLTVPPCSEGVTWILFRYPLTVSQLQIEEFRRLRTHVKGTEL-LDSCDGILGDNFRPTQPLSDRIIRAAFQ : 257 CAH8_Xtro : KGKSKTIPC-FNPNTLLP---DPLLRDYWVYEGSLTMPPCSEGVTWILFRYPLTVSQTQIEEFRRLRTHIKGADL-PDGCDGLMADNFRPTQPLSDRIIRAAFQ : 282 CAH8_Spur : KGRTLAVTTPFHPSCLLP---DPQLRDFWTYQGSLTTPPCYERVTWILLRYPLTISISQMEEFRRLKNHVKGDFS-FRGEEGLLVDNFRPTQPLNDRTIEASFQ : 312 CAH8_Blan : KGKSKTITTAFNPAAFLP---DPALREYWTYEGSLTSPPCYENVQWIIFRYPLTISHVQMEEFRRLRCHAKGQPP-PPGEEGMLCDNFRPTQPIKSRVIRASFQ : 256 CAH8_Tadh : KDDIREMRLPFNPIHLVP-----RIRDYWTYDGSLTIPPCTENVTWIIFRYPLSISESQIQGYRSLRAFFRQDRQDVASREGALFDNFRPVQSMNNRPLRASFE : 259

Figure 1 Multiple sequence alignment of CARP VIII sequences. Comparison of 33 CARP VIII sequences by multiple sequence alignment. Short names (first letter of the genus and first three letters of the species) are provided on the left side and residue numbers are provided on the right side of the figure. Details of the sequences and full species names are provided in Table 1. The sequences of CAH8_Rnor and CAH8_Spur are trimmed in the end by 21 and 89 residues, respectively. Aspatwar et al. BMC Molecular Biology 2010, 11:25 Page 6 of 19 http://www.biomedcentral.com/1471-2199/11/25

* 20 * 40 * 60 * 80 * 100 * CAH10b_Tni : MHTVWDIVVILQTFLICTSSAQPITSKLDDSWWAYKDVVQGSFVPVPSFWGLVHSAWNLCAIGKRQSPINIETSHMIFDPYLTPLKLNTGGRKMGGTMYNTGKHVSLRPDK : 111 CAH10b_Tru : MPNVWDIAVVLQTFIICTSSAQPIASKLDDSWWAYKDVVQGSFVPVPSFWGLVHSAWNLCAIGKRQSPINIETSHMIFDPYLTPLKLNTGGRKMGGTMYNTGKHVSLRPDK : 111 CAH10b_Gac : MHLVWDLSVILQTFLICTSSAQPLTAKLDAGWWAYKDVVHGSFVPVPSFWGLVHSAWNLCAIGKKQSPIDIDTSRLIFDPYLTPLRLNTAGQKMGCTMYNTGKHVSLRPDK : 111 CAH10b_Dre : ------TAQPVSSKLHDGWWAYKDVVQGSFIPVPSFWGLVNTAWNLCAIGKRQSPVNIETSRMIFDPFLNPLRLNAGQRKVSGTMYNTGRHVSLRPDK : 92 CAH10a_Gac : MDIFWEILIILHANVIVCISAQPNPP-----WWAYKEVVQGSFVPVPSFWGLVNSAWNLCSVGKRQSPVNIETSHMIFDPFLQPIKLNTGGRKVGGTMYNTGRHVSLRLDK : 106 CAH10a_Tru : ------WWAYKEVVQGSFVPVPSFWGLVNSAWNLCSVGKRQSPVNIETSHMIFDPFLLPIKLNTGGHKVGGTMYNTGRHVSLRLDK : 80 CAH10a_Dre : MNIIWEIFIILQANLIVCSSAQPNPPKIHEGWWAYKEVVQGSFVPVPSFWGLVNSAWNLCSVGKRQSPVNIETSHMIFDPFLTPLRLNTGGRKVGGTMYNTGRHVSLRLDK : 111 CAH10_Acar : MEIVWEVLFLLQANFIVCISAQQNSPKIHEGWWAYKEVVQGSFVPVPSFWGLVNSAWNLCSVGKRQSPVNIETSHMIFDPFLTPLRINTGGRKVSGTMYNTGRHVSLRLDK : 111 CAH10_Ggal : MEIVWEVLFLLQANFIVCISAQQNSPKIHEGWWAYKEVVQGSFVPVPSFWGLVNSAWNLCSVGKRQSPVNIETSHMIFDPFLTPLRINTGGRKVSGTMYNTGRHVSLRLDK : 111 CAH10_Tgut : MEIVWEVLFLLQANFIVCISAQQNSPKIHEGWWAYKEVVQGSFVPVPSFWGLVNSAWNLCSVGKRQSPVNIETSHMIFDPFLTPLRLNTGGRKVSGTMYNTGRHVSLRLDK : 111 CAH10_Mdom : MEIVWEVLFLLQANFIVCISAQQNSPKIHEGWWAYKEVVQGSFVPVPSFWGLVNSAWNLCSVGKRQSPVNIETSHMIFDPFLTPLRINTGGRKVSGTMYNTGRHVSLRLDK : 111 CAH10_Ocun : ------AQQNSPKIHEGWWAYKEVVQGSFVPVPSFWGLVNSAWNLCSVGKRQSPVNIETSHMIFDPFLTPLRINTGGRKVSGTMYNTGRHVSLRLDK : 91 CAH10_Ttru : ------AQQNSPKIHEGWWAYKEVVQGSFVPVPSFWGLVNSAWNLCSVGKRQSPVNIETSHMIFDPFLTPLRINTGGRKVSGTMYNTGRHVSLRLDK : 91 CAH10_Rnor : MEIVWEVLFLLQANFIVCISAQQNSPKIHEGWWAYKEVVQGSFVPVPSFWGLVNSAWNLCSVGKRQSPVNIETSHMIFDPFLTPLRINTGGRKVSGTMYNTGRHVSLRLDK : 111 CAH10_Cfam : MEIVWEVLFLLQANFIVCISAQQNSPKIHEGWWAYKEVVQGSFVPVPSFWGLVNSAWNLCSVGKRQSPVNIETSHMIFDPFLTPLRINTGGRKVSGTMYNTGRHVSLRLDK : 111 CAH10_Cpor : MEIVWEVLFLLQANFIVCISAQQNSPKIHEGWWAYKEVVQGSFVPVPSFWGLVNSAWNLCSVGKRQSPVNIETSHMIFDPFLTPLRINTGGRKVSGTMYNTGRHVSLRLDK : 111 CAH10_Mmul : MEIVWEVLFLLQANFIVCISAQQNSPKIHEGWWAYKEVVQGSFVPVPSFWGLVNSAWNLCSVGKRQSPVNIETSHMIFDPFLTPLRINTGGRKVSGTMYNTGRHVSLRLDK : 111 CAH10_Ecab : MEIVWEVLFLLQANFIVCISAQQNSPKIHEGWWAYKEVVQGSFVPVPSFWGLVNSAWNLCSVGKRQSPVNIETTHMIFDPFLTPLRINTGGRKVSGTMYNTGRHVSLRLDK : 111 CAH10_Dnov : MEIVWEVLFLLQANFIVCISAQQNSPKIHEGWWAYKEVVQGSFVPVPSFWGLVNSAWNLCSVGKRQSPVNIETSHMIFDPFLTPLRINTGGRKVSGTMYNTGRHVSLRLDK : 111 CAH10_Btau : MEIVWEVLFLLQANFIVCISAQQNSPKIHEGWWAYKEVVQGSFVPVPSFWGLVNSAWNLCSVGKRQSPVNIETSHMIFDPFLTPLRINTGGRKVSGTMYNTGRHVSLRLDK : 111 CAH10_Mmus : MEIVWEVLFLLQANFIVCISAQQNSPKIHEGWWAYKEVVQGSFVPVPSFWGLVNSAWNLCSVGKRQSPVNIETSHMIFDPFLTPLRINTGGRKVSGTMYNTGRHVSLRLDK : 111 CAH10_Mfas : MEIVWEVLFLLQANFIVCISAQQNSPKIHEGWWAYKEVVQGSFVPVPSFWGLVNSAWNLCSVGKRQSPVNIETSHMIFDPFLTPLRINTGGRKVSGTMYNTGRHVSLRLDK : 111 CAH10_Pabe : MEIVWEVLFLLQANFIVCISAQQNSPKIHEGWWAYKEVVQGSFVPVPSFWGLVNSAWNLCSVGKRQSPVNIETSHMIFDPFLTPLRINTGGRKVSGTMYNTGRHVSLRLDK : 111 CAH10_Ogar : MEIVWEVLFLLQANFIVCISAQQNSPKIHEGWWAYKEVVQGSFVPVPSFWGLVNSAWNLCSVGKRQSPVNIETSHMIFDPFLTPLRINTGGRKVSGTMYNTGRHVSLRLDK : 111 CAH10_Fcat : MEIVWEVLFLLQANFIVCIS-QQNSPKIHEGWWAYKEVVQGSFVPVPSFWGLVNSAWNLCSVGKRQSPVNIETSHMIFDPFLTPLRINTGGRKVSGTMYNTGRHVSLRLDK : 110 CAH10_Hsap : MEIVWEVLFLLQANFIVCISAQQNSPKIHEGWWAYKEVVQGSFVPVPSFWGLVNSAWNLCSVGKRQSPVNIETSHMIFDPFLTPLRINTGGRKVSGTMYNTGRHVSLRLDK : 111 CAH10_Cjac : MEIVWEVLFLLQANFIVCISAQQNSPKIHEGWWAYKEVVQGSFVPVPSFWGLVNSAWNLCSVGKRQSPVNIETSHMIFDPFLTPLRINTGGRKVSGTMYNTGRHVSLRLDK : 111 CAH10_Ppyg : MEIVWEVLFLLQANFIVCISAQQNSPKIHEGWWAYKEVVQGSFVPVPSFWGLVNSAWNLCSVGKRQSPVNIETSHMIFDPFLTPLRINTGGRKVSGTMYNTGRHVSLRLDK : 111 CAH10_Ptro : MEIVWEVLFLLQANFIVCISAQQNSPKIHEGWWAYKEVVQGSFVPVPSFWGLVNSAWNLCSVGKRQSPVNIETSHMIFDPFLTPLRINTGGRKVSGTMYNTGRHVSLRLDK : 111 CAH10_Oana : ------AQQNSPKIHEGWWAYKEVVQGSFVPVPSFWGLVNSAWNLCSVGKRQSPVNIETSHMIFDPFLTPLRINTGGRKVGGTMYNTGRHVSLRLDK : 91 CAH10_Xtro : MEIVWEVFFILQASLIVCMSAQQNSPKIHEGWWTYKEVVQGSFVPVPSFWGLVNSAWNLCAVGKRQSPVNIETSHMIFDPFLTPLRINTGGRKVSGTMYNTGRHVSLRLDK : 111 CAH10_Blan : ---MIPYVLLTVALVQQLSTNAEGQAVAWDDWWTYEGITG------PQFWGLVNSDWVMCKDGKMQSPINFRANQILYDPNLIPINFDSDTDKMNGTLLNTGHDLTFQVDD : 102 120 * 140 * 160 * 180 * 200 * 220 CAH10b_Tni : AHLVNISGGPLGYSYRLEEVRLHFGSEDSQGSEHLLNGQGFPGEVQLIHYNQDLYANYTEAAKSPHGIAVVSIFIKLSENSNSFLNRMLNRDTITRINNRHDAFLLMGLNI : 222 CAH10b_Tru : AHLVNISGGPLGYSYRLEEVRLHFGSEDSQGSEHLLNGQGFPGEVQLIHYNQDLYANYSEAAKSPHGIAVVSIFIKLSENSNSFLNRMLNRDTITRINYRHDAFLLMGLNI : 222 CAH10b_Gac : AHLVNISGGPLGYSYRLEEVRLHFGSEDSQGSEHLLNGQGFTGEVQLIHYNQDLYGNYTEAAKSPHGIAVVSIFMKLSENSNAFLNRMLHRDSITRINYKHDAFLLMGLNI : 222 CAH10b_Dre : SHLVNISGGPLSYSYRLEEIRLHFGSEDNRGSEHLLNGQAFPGEVQLIHYNQDLYLNYSDAVRSPNGIAVVSIFMKISEPTNVFLNRMLNRETVTRITYKHDAYLLMGLNI : 203 CAH10a_Gac : EHLVNISGGPMTYSHRLEEIRLHFGSEDGQGSEHLLNGQAFSGEVQLIHYNHELYTNYTEAAKSPNGLVIVSIFMKIAETANSFLNRMLNRDTITRITYKNDAYLLTNLNI : 217 CAH10a_Tru : EHLVNISGGPMTYSHRLEEIRLHFGSEDGQGSEHLLNGQAFSGEVQLIHYNHELYTNYTEAAKSPNGLVIISIFMKIAETSNSFLNRMLNRDTITRITYKNDAYLLTGLNI : 191 CAH10a_Dre : EHLVNISGGPMTYSHRLEEIRLHFGSEDGQGSEHLLNGQAFSGEVQLIHYNHELYTNYTEAAKSPNGLVIVSIFMKISETSNSFLNRMLNRDTITRITYKNDAYLLSGLNI : 222 CAH10_Acar : EHLVNISGGPMTYSHRLEEIRLHFGSEDSQGSEHLLNGQAFSGEVQLIHYNHELYTNVTEAAKSPNGLVVVSIFMKVSESSNPFLNRMLNRDTITRITYKNDAYLLQGLNI : 222 CAH10_Ggal : EHLVNISGGPMTYSHRLEEIRLHFGSEDSQGSEHLLNGQAFSGEVQLIHYNHELYTNVTEAAKSPNGLVVVSIFMKVSESSNPFLNRMLNRDTITRITYKNDAYLLQGLNI : 222 CAH10_Tgut : EHLVNISGGPMTYSHRLEEIRLHFGSEDSQGSEHLLNGQAFSGEVQLIHYNHELYTNVTEAAKSPNGLVVVSIFMKVSESSNPFLNRMLNRDTITRITYKNDAYLLQGLNI : 222 CAH10_Mdom : EHLVNISGGPMTYSHRLEEIRLHFGSEDSQGSEHLLNGQAFSGEVQLIHYNHELYTNVTEAAKSPNGLVVVSIFMKVSDSSNPFLNRMLNRDTITRITYKNDAYLLQGLNI : 222 CAH10_Ocun : EHLVNISGGPMTYSHRLEEIRLHFGSEDSQGSEHLLNGQAFSGEVQLIHYNHELYTNVTEAAKSPNGLVVVSIFIKVSDSSNPFLNRMLNECTITRITYKNDAYLLQGLNI : 202 CAH10_Ttru : EHLVNVSGGPMTYSHRLEEVRLHFGSEDSQGSEHLLNGQAFSGEVQLIHYNHELYTNVTEAAKSPNGLVVVSIFIKVSDSSNPFLNRMLNRDTITRITYKNDAYLLQGLNI : 202 CAH10_Rnor : EHLVNISGGPMTYSHRLEEIRLHFGSEDSQGSEHLLNGQAFSGEVQLIHYNHELYTNVTEAAKSPNGLVVVSIFIKVSDSSNPFLNRMLNRDTITRITYKNDAYLLQGLNI : 222 CAH10_Cfam : EHLVNISGGPMTYSHRLEEIRLHFGSEDSQGSEHLLNGQAFSGEVQLIHYNHELYTNVTEAAKSPNGLVVVSIFIKVSDSSNPFLNRMLNRDTITRITYKNDAYLLQGLNI : 222 CAH10_Cpor : EHLVNISGGPMTYSHRLEEIRLHFGSEDSQGSEHLLNGQAFSGEVQLIHYNHELYTNVTEAAKSPNGLVVVSIFIKVSDSSNPFLNRMLNRDTITRITYKNDAYLLQGLNI : 222 CAH10_Mmul : EHLVNISGGPMTYSHRLEEIRLHFGSEDSQGSEHLLNGQAFSGEVQLIHYNHELYTNVTEAAKSPNGLVVVSIFIKVSDSSNPFLNRMLNRDTITRITYKNDAYLLQGLNI : 222 CAH10_Ecab : EHLVNISGGPMTYSHRLEEIRLHFGSEDSQGSEHLLNGQAFSGEVQLIHYNHELYTNVTEAAKSPNGLVVVSIFIKVSDSSNPFLNRMLNRDTITRITYKNDAYLLQGLNI : 222 CAH10_Dnov : EHLVNISGGPMTYSHRLEEIRLHFGSEDSQGSEHLLNGQAFSGEVQLIHYNHELYTNVTEAAKSPNGLVVVSIFIKVSDSSNPFLNRMLNRDTITRITYKNDAYLLQGLNI : 222 CAH10_Btau : EHLVNISGGPMTYSHRLEEIRLHFGSEDSQGSEHLLNGQAFSGEVQLIHYNHELYTNVTEAAKSPNGLVVVSIFIKVSDSSNPFLNRMLNRDTITRITYKNDAYLLQGLNI : 222 CAH10_Mmus : EHLVNISGGPMTYSHRLEEIRLHFGSEDSQGSEHLLNGQAFSGEVQLIHYNHELYTNVTEAAKSPNGLVVVSIFIKVSDSSNPFLNRMLNRDTITRITYKNDAYLLQGLNI : 222 CAH10_Mfas : EHLVNISGGPMTYSHRLEEIRLHFGSEDSQGSEHLLNGQAFSGEVQLIHYNHELYTNVTEAAKSPNGLVVVSIFIKVSDSSNPFLNRMLNRDTITRITYKNDAYLLQGLNI : 222 CAH10_Pabe : EHLVNISGGPMTYSHRLEEIRLHFGSEDSQGSEHLLNGQAFSGEVQLIHYNHELYTNVTEAAKSPNGLVVVSIFIKVSDSSNPFLNRMLNRDTITRITYKNDAYLLQGLNI : 222 CAH10_Ogar : EHLVNISGGPMTYSHRLEEIRLHFGSEDSQGSEHLLNGQAFSGEVQLIHYS-ELYTNVTEAAKSPNGLVVVSIFIKVSDSSNPFLNRMLNRDTITRITYKNDAYLLQGLNI : 221 CAH10_Fcat : EHLVNISGGPMTYSHRLEEIRLHFGSEDSQGSEHLLNGQAFSGEVQLIHYNHELYTNVTEAAKSPNGLVVVSIFIKVSDSSNPFLNRMLNRDTITRITYKNDAYLLQGLNI : 221 CAH10_Hsap : EHLVNISGGPMTYSHRLEEIRLHFGSEDSQGSEHLLNGQAFSGEVQLIHYNHELYTNVTEAAKSPNGLVVVSIFIKVSDSSNPFLNRMLNRDTITRITYKNDAYLLQGLNI : 222 CAH10_Cjac : EHLVNISGGPMTYSHRLEEIRLHFGSEDSQGSEHLLNGQAFSGEVQLIHYNHELYTNVTEAAKSPNGLVVVSIFIKVSDSSNPFLNRMLNRDTITRITYKNDAYLLQGLNI : 222 CAH10_Ppyg : EHLVNISGGPMTYSHRLEEIRLHFGSEDSQGSEHLLNGQAFSGEVQLIHYNHELYTNVTEAAKSPNGLVVVSIFIKVSDSSNPFLNRMLNRDTITRITYKNDAYLLQGLNI : 222 CAH10_Ptro : EHLVNISGGPMTYSHRLEEIRLHFGSEDSQGSEHLLNGQAFSGEVQLIHYNHELYTNVTEAAKSPNGLVVVSIFIKVSDSSNPFLNRMLNRDTITRITYKNDAYLLQGLNI : 222 CAH10_Oana : EQLVNISGGPMTYSHRLEEIRLHFGSEDSQGSEHLLNGQAFSGEVQLIHYNHELYTNVTEAAKSPNGLVVVSIFMKVSDSSNPFLNRMLNRDTITRITYKNDAYLLQGLNI : 202 CAH10_Xtro : EHLVNISGGPLTYSHRLEEIRLHFGSEDGQGSEHLLNGQAFSGEVQLIHYNHELYTNVTDAAKSPNGLVVISIFIKVTDSSNLFLNRMLNRDTITRITYKNDAYLLQGLNI : 222 CAH10_Blan : RNRVNLTGGPLSYLYRLQELRLHYGSIDSQGSEHSVDGYSFPGELQLLFYNYDLYGDLAEATKGTSGLAIISLFIQIGNSTNPELDKLLWSDRLQNVTYKGSAARVEDFNL : 213 * 240 * 260 * 280 * 300 * 320 * CAH10b_Tni : ADLYPDTTRYITYEGSITIPPCYETSTWILINKPVYVTQMQMHSLRLLSQNEPYKIFLSMSDNIRPAQPLLQRCIRTNINF---SKQGRNCPNNRALRPQYRVNQWLLK : 328 CAH10b_Tru : ADLYPDTTRYITYEGSITIPPCYETSTWILINKPVYVTQMQMHSLRLLSQNEPYKIFLSMSDNIRPTQPLLQRCIRTNINF---SKQGRNCPNNRALRPQYRVNQWLLK : 328 CAH10b_Gac : ADLYPDTTRYITYEGSITVPPCYETSTWILINKPVYVTRMQMHSLRLLSQNEPYKIFLSMSDNTRPPQPLLQRCIRTNINF---NKPGRDCPNNRVLRPQYRVNQWLLK : 328 CAH10b_Dre : EELYPETSRFITYEGSITIPPCLETATWILMNKPIYISQIEMQSLRLLSQNQPSQIFLSMGDNMRPTQTLHQRCIRTNINF---SQR-RDCPNNRMLRPQYRVNEWLLK : 308 CAH10a_Gac : EEIYPETISFITYEGSMTIPPCFETATWILMNKPVYLTRMQMHSLRLLSQNQPSQIFLSMSDNVRPVQPLNNRCIRTNINF---SMQGKDCPNNRAQKLQYRVNEWLLK : 323 CAH10a_Tru : EEIYPDTSSFITYDGSMTIPPCFETVTWILMNKPVYLTRMQMHSLRLLSQNLPSQIFLSMSDNVRPVQPLNNRCIRTNINF---SMQGKDCPNNRAQKLQYRVNEWLLK : 297 CAH10a_Dre : EEVYPETASFITYEGSMTIPPCYETATWILMNKPIYVTKMQMHSMRLLSQNQPSQIFLSMSDNVRPVQPLNNRCIRTNINF---SMQGKDCPNNRAQKLQYRVNEWLLK : 328 CAH10_Acar : EELYPETASFITYDGSMTIPPCYETASWILMNKPVYITRMQMHSLRLLSQNQPSQIFLSMSDNFRPVQPLNNRCIRTNINF---SLQGKDCPNNRAQKLQYR------: 321 CAH10_Ggal : EELYPETSSFITYDGSMTIPPCYETASWIIMNKPVYITRMQMHSLRLLSQNQPSQIFLSMSDNFRPVQPLNNRCIRTNINF---SLQGKDCPNNRAQKLQYRVNEWLLK : 328 CAH10_Tgut : EELYPETSSFITYDGSMTIPPCYETASWIIMNKPVYITRMQMHSLRLLSQNQPSQIFLSMSDNFRPVQPLNNRCIRTNINF---SLQGKDCPNNRAQKLQYRVNEWLLK : 328 CAH10_Mdom : EELYPETSSFITYDGSMTIPPCYETASWIIMNKPVYITRMQMHSLRLLSQNQPSQIFLSMSDNFRPVQPLNNRCIRTNINF---SLQGKDCPNNRAQKLQYRVNEWLLK : 328 CAH10_Ocun : EELYPETSSFITYDGSMTIPPCYETASWIIMNKPVYITRMQMHSLRLLSQNQPSQIFLSMSDNFRPVQPLNNRCIRTNINF---SLQGKDCPNNRAQKLQYRVNEWLLK : 308 CAH10_Ttru : EELYPETSSFITYDGSMTIPPCYETASWIIMNKPVYITRMQMHSLRLLSQNQPSQIFLSMSDNFRPVQPLNNRCIRTNINF---SLQGKDCPNNRAQKLQYRVNEWLLK : 308 CAH10_Rnor : EELYPETSSFITYDGSMTIPPCYETASWIIMNKPVYITRMQMHSLRLLSQNQPSQIFLSMSDNFRPVQPLNNRCIRTNINF---SLQGKDCPNNRAQKLQYRVNEWLLK : 328 CAH10_Cfam : EELYPETSSFITYDGSMTIPPCYETASWIIMNKPVYITRMQMHSLRLLSQNQPSQIFLSMSDNFRPVQPLNNRCIRTNINF---SLQGKDCPNNRAQKLQYRVNEWLLK : 328 CAH10_Cpor : EELYPETSSFITYDGSMTIPPCYETASWIIMNKPVYITRMQMHSLRLLSQNQPSQIFLSMSDNFRPVQPLNNRCIRTNINF---SLQGKDCPNNRAQKLQYRVNEWLLK : 328 CAH10_Mmul : EELYPETSSFITYDGSMTIPPCYETASWIIMNKPVYITRMQMHSLRLLSQNQPSQIFLSMSDNFRPVQPLNNRCIRTNINF---SLQGKDCPNNRAQKLQYR------: 321 CAH10_Ecab : EALYPETSSFITYDGSMTIPPCYETASWIIMNKPVYITRMQMHSLRLLSQNQPSQIFLSMSDNFRPVQPLNNRCIRTNINF---SLQGKDCPNNRAQKLQYRVNEWLLK : 328 CAH10_Dnov : EGTYPGTSSFINYDESMTIPPCYETAGWIIMN-PMCITRMQIHSLCLLSQNQPSQIVPNMSDNFRPVRPLNNCCIHPNINF---SLQGKDCPNNRAQELQYRINEWLLK : 327 CAH10_Btau : EELYPETSSFITYDGSMTIPPCYETANWIIMNKPVYITRMQMHSLRLLSQNQPSQIFLSMSDNFRPVQSLNNRCIRTNINF---SLQGKDCPNNRAQKLQYRVNEWLLK : 328 CAH10_Mmus : EELYPETSSFITYDGSMTIPPCYETASWIIMNKPVYITRMQMHSLRLLSQNQPSQIFLSMSDNFRPVQPLNNRCIRTNINF---SLQGKDCPNNRAQKLQYRVNEWLLK : 328 CAH10_Mfas : EELYPETSSFITYDGSMTIPPCYETASWIIMNKPVYITRMQMHSLRLLSQNQPSQIFLSMSDNFRPVQPLNNRCIRTNINF---SLQGKDCPNNRAQKLQYRVNEWLLK : 328 CAH10_Pabe : EELYPETSSFITYDGSMTIPPCYETASWIIMNKPVYITRMQMHSLRLLSQNQPSQIFLSMSDNFRPVQPLNNRCIRTNINF---SLQGKDCPNNRAQKLQYRVNEWLLK : 328 CAH10_Ogar : EELYPETSSFITYDGSMTIPPCYETASWIIMNKPVYITRMQMHSLRLLSQNQPSQIFLSMSDNFRPVQPLNNRCIRTNINF---SLQGKDCPNNRAQKLQYRVNEWLLK : 327 CAH10_Fcat : EELYPETSSFITYDGSMTIPPCYETASWIIMNKPVYITRMQMS-LRLLSQNQPSQIFLSMSDNFRPVQPLNNRCIRTNINF---SLQGKDCPNNRAQKLQYRVNEWLLK : 326 CAH10_Hsap : EELYPETSSFITYDGSMTIPPCYETASWIIMNKPVYITRMQMHSLRLLSQNQPSQIFLSMSDNFRPVQPLNNRCIRTNINF---SLQGKDCPNNRAQKLQYRVNEWLLK : 328 CAH10_Cjac : EELYPETSSFITYDGSMTIPPCYETASWIIMNKPVYITRMQMHSLRLLSQNQPSQIFLSMSDNFRPVQPLNNRCIRTNINF---SLQGKDCPNNRAQKLQYRVNEWLLK : 328 CAH10_Ppyg : EELYPETSSFITYDGSMTIPPCYETASWIIMNKPVYITRMQMHSLRLLSQNQPSQIFLSMSDNFRPVQPLNNRCIRTNINF---SLQGKDCPNNRAQKLQYRVNEWLLK : 328 CAH10_Ptro : EELYPETSSFITYDGSMTIPPCYETASWIIMNKPVYITRMQMHSLRLLSQNQPSQIFLSMSDNFRPVQPLNNRCIRTNINF---SLQGKDCPNNRAQKLQYRVNEWLLK : 328 CAH10_Oana : EELYPETASFITYDGSMTTPPCYETASWIIMNKPVYITRMQMHSLRLLSQNQPSQIFLSMSDNFRPGQPLNNRCIRTNINF---SLQGKDCPNHRPQKLQYRVNEWLLK : 308 CAH10_Xtro : EEIYPETSSFITYDGSMTIPPCYETASWIIMNKPIYITRMQMHSLRLLSQNQPSQIFSSMSDNFRPVQSLNNRCIRTNINF---RLQGKDCPNNRAQKLQYK------: 321 CAH10_Blan : TNLFPTTPHYMTYQGSLTSPDCAETVTWIVMNKPVYISRKKMNQLRNLWQNTMGQKETPLADNFRPTSNLNSRTIRTNINFTTKDKMGKPCPTMKSKKAYQSSNRC--- : 319

Figure 2 Multiple sequence alignment of CARP X sequences. Comparison of 32 CARP X sequences by multiple sequence alignment. Short names (first letter of the genus and first three letters of the species) are provided on the left side and residue numbers are provided on the right. Details of the sequences and full species names are provided in Table 1. Aspatwar et al. BMC Molecular Biology 2010, 11:25 Page 7 of 19 http://www.biomedcentral.com/1471-2199/11/25

* 20 * 40 * 60 * 80 CAH11_Rnor : MGGAARLSAPQALVLWAALGAAAHIGPAPDPEDWWSYKENLQGNFVPGPPFWGLVNAAWSLCAVGKRQSPVDVELKRVLYDPFLP : 85 CAH11_Mmus : MGGAARLSAPQALVLWAALGAAAHIGPAPDPEDWWSYKENLQGNFVPGPPFWGLVNAAWSLCAVGKRQSPVDVELKRVLYDPFLP : 85 CAH11_Fcat : MGVAARLSAPRALVLWAALGAAAHIGPAPDPEDWWSYKDNLQGNFVPGPPFWGLVNAAWSLCAVGKRQSPVDVELKRVLYDPFLP : 85 CAH11_Ecab : MGGAAPLSAPQALVLWAALGAAAYIGPAPDPEDWWSYKDNLQGNFVPGPPFWGLVNAAWSLCAVGKRQSPVDVELKRVLYDPFLP : 85 CAH11_Sscr : MGGAARLSAPRALVLWAVLGAAAHIGPAPDPEDWWSYKDNLQGNFVPGPPFWGLVNAAWSLCAVGKRQSPVDVELKRVLYDPFLP : 85 CAH11_Btau : MGAAARLSAPRALVLWAALGAAAHIGPAPDPEDWWSYKDNLQGNFVPGPPFWGLVNAAWSLCAVGKRQSPVDVELKRVLYDPFLP : 85 CAH11_Cfam : MGAAARLSAPRALVLWAALGAAAHIGPAPDPEDWWSYKDNLQGNFVPGPPFWGLVNAAWSLCAVGKRQSPVDVELKRVLYDPFLP : 85 CAH11_Hsap : MGAAARLSAPRALVLWAALGAAAHIGPAPDPEDWWSYKDNLQGNFVPGPPFWGLVNAAWSLCAVGKRQSPVDVELKRVLYDPFLP : 85 CAH11_Ptro : MGAAARLSAPRALVLWAALGAAAHIGPAPDPEDWWSYKDNLQGNFVPGPPFWGLVNAAWSLCAVGKRQSPVDVELKRVLYDPFLP : 85 CAH11_Pabe : MGAAARLSAPRALVLWAALGAAAHIGPAPDPEDWWSYKDNLQGNFVPGPPFWGLVNAAWSLCAVGKRQSPVDVELKRVLYDPFLP : 85 CAH11_Ppyg : MGAAARLSAPRALVLWAALGAAAHIGPAPDPEDWWSYKDNLQGNFVPGPPFWGLVNAAWSLCAVGKRQSPVDVELKRVLYDPFLP : 85 CAH11_Mmul : MGAAARLSAPRALVLWAALGAAAHIGPAPDPEDWWSYKDNLQGNFVPGPPFWGLVNAAWSLCAVGKRQSPVDVELKRVLYDPFLP : 85 CAH11_Cjac : MGAAARLSAPRALVLWAALGAAAHIGPAPDPEDWWSYKDNLHGNFVPGPPFWGLVNAAWSLCAVGKRQSPVDVELKRVLYDPFLP : 85 CAH11_Ttru : MGGAARLNAPRALLLWAALGAAAHIGPAPDPEDWWSYKDNLQGNFVPGPPFWGLVNAAWSLCAVGKRQSPVDVELKRVLYDPFLP : 85 CAH11_Oari : MGAAPRLSAPRVLVLWAALGAAAHIGPAPDPEDWWSYKDNLQGNFVPGPPFWGLVNAAWSLCAVGKRQSPVDVDLKRVLYDPFLP : 85 CAH11_Cpor : ------AHIGPAPDPEDWWSYKDNLQGNFVPGPPFWGLVNAAWSLCAVGKRQSPVDVDLKRVLYDPFLP : 63 CAH11_Mdom : MGGPMRLSPPRALLLGAALGAAAHIGPAPAPEDWWTYKDNLQGSFVPGPPFWGLVNAAWSLCAVGKRQSPVDIELRRVLYDPFLP : 85 CAH11_Acar : ------EDWWAYKETLHGSFVPGPPFWGLVNSAWSLCAIGKRQSPVDVNTSQMVYDPFLP : 54 CAH11_Xtro : ----RRVIYP------GESAQFSPAKTYEEWWAYKEAVQGSFVPGPAFWGLVNSAWNLCTIGKRQSPININTSQIIFDPFLP : 72 * 100 * 120 * 140 * 160 * CAH11_Rnor : PLRLSTGGEKLRGTLYNTGRHVSFLPASRPVVNVSGGPLLYSHRLSELRLLFGARDGAGSEHQINHQGFSAEV-QLIHFNQELYG : 169 CAH11_Mmus : PLRLSTGGEKLRGTLYNTGRHVSFLPASRPVVNVSGGPLLYSHRLSELRLLFGARDGAGSEHQINHEGFSAEV-QLIHFNQELYG : 169 CAH11_Fcat : PLRLSTGGEKLRGTLYNTGRHVSFLPAPRPVVNVSGGPLLYSHRLSELRLLFGARDGAGSEHQINHQGFSAEV-QLIHFNQELYG : 169 CAH11_Ecab : PLRLSTGGEKLRGTLYNTGRHVSFLPAARPVVNVSGGPLLYSHRLSELRLLFGARDGAGSEHQINHQGFSAEV-QLIHFNQELYG : 169 CAH11_Sscr : PLRLSTGGEKLRGTLYNTGRHVSFLPAPRPVVNVSGGPLLYSHRLSELLLLFGAHDGAGSEHQINHQGFSAEV-QLIHFNQELYG : 169 CAH11_Btau : PLRLSTGGEKLRGTLYNTGRHVSFLPAPRPVVNVSGGPLLYSHRLSELRLLFGARDGAGSEHQINHQGFSAEV-QLIHFNQELYG : 169 CAH11_Cfam : PLRLSTGGEKLRGTLYNTGRHVSFLPAPRPVVNVSGGPLLYSHRLSELRLLFGARDGAGSEHQINHQGFSAEV-QLIHFNQELYG : 169 CAH11_Hsap : PLRLSTGGEKLRGTLYNTGRHVSFLPAPRPVVNVSGGPLLYSHRLSELRLLFGARDGAGSEHQINHQGFSAEV-QLIHFNQELYG : 169 CAH11_Ptro : PLRLSTGGEKLRGTLYNTGRHVSFLPAPRPVVNVSGGPLLYSHRLSELRLLFGARDGAGSEHQINHQGFSAEV-QLIHFNQELYG : 169 CAH11_Pabe : PLRLSTGGEKLRGTLYNTGRHVSFLPAPRPVVNVSGGPLLYSHRLSELRLLFGARDGAGSEHQINHQGFSAEV-QLIHFNQELYG : 169 CAH11_Ppyg : PLRLSTGGEKLRGTLYNTGRHVSFLPAPRPVVNVSGGPLLYSHRLSELRLLFGARDGAGSEHQINHQGFSAEV-QLIHFNQELYG : 169 CAH11_Mmul : PLRLSTGGEKLRGTLYNTGRHVSFLPAPRPVVNVSGGPLLYSHRLSELRLLFGARDGAGSEHQINHQGFSAEV-QLIHFNQELYG : 169 CAH11_Cjac : PLRLSTGGEKLRGTLYNTGRHVSFLPAPRPVVNVSGGPLLYSHRLSELRLLFGARDGAGSEHQINHQGFSAEVVQLIHFNQELYG : 170 CAH11_Ttru : PLRLSTGGEKLRGTLYNTGRHVSFLPAPRPVVNVSGGPLLYSHRLSELRLLFGARDGAGSEHQINHQGFSAEV-QLIHFNQELYG : 169 CAH11_Oari : PLRLSTGGEKLRGTLYNTGRHVSFLPAPRPVVNVSGGPLLYSHRLSELRLLFGARDGAGSEHQINHQGFSAEV-QLIHFNQELYG : 169 CAH11_Cpor : PLRLSTGGEKLRGTLYNTGRHVSFLPASRPVVNVSGGPLLYSHRLSELRLLFGARDGAGSEHQINHQGFSAEV-QLIHFNQELYG : 147 CAH11_Mdom : PLRLSTGGEKLRGTLYNTGRHVSFLPAPRPVVNVSGGPLLYSHRLSELRLLFGARDGAGSEHQINHESFSAEV-QLIHFNQELYG : 169 CAH11_Acar : PLRLSTGGKKISGTMYNTGRHVSFRPDPLQLVNVSGGPLLYSHRLQEIRLHFGSEDGFGSEHQIDGETYSAEV-QLIHYNQELYP : 138 CAH11_Xtro : PLRLSMTGTTVSGTMHNTGRHVSFRLDKEQPVNISGGPLLYNHRLEEVMLHFGSENSIGSEHLMNKETSAGEV-QLIHYNQDLYS : 156 180 * 200 * 220 * 240 * CAH11_Rnor : NLSAASRGPNGLAILSLFVN------VAGSSNPFLSRLLNRDTITRISYKNDAYFLQDLSLELLFPESFGFITYQGSLSTPPCSE : 248 CAH11_Mmus : NLSAASRGPNGLAILSLFVN------VAGSSNPFLSRLLNRDTITRISYKNDAYFLQDLSLELLFPESFGFITYQGSLSTPPCSE : 248 CAH11_Fcat : NLSAASRGPNGLAILSLFVN------VAGSSNPFLSRLLNRDTITRISYKNDAYFLQDLSLELLFPESFGFITYQGSLSTPPCSE : 248 CAH11_Ecab : NLSAASRGPNGLAILSLFVN------VAGSSNPFLSRLLNRDTITRISYKNDAYFLQDLSLELLFPESFGFITYQGSLSTPPCSE : 248 CAH11_Sscr : NLSAASRGPNGLAILSLFVNV---SQVAGNSNPFLSRLLNRDTITRISYKNDAYFLQDLSLELLFPESFGFITYQGSLSTPPCSE : 251 CAH11_Btau : NLSAATRGPNGLAILSLFVN------VAGSSNPFLSRLLNRDTITRISYKNDAYFLQDLSLELLFPESFGFITYQGSLSTPPCSE : 248 CAH11_Cfam : NLSAASRGPNGLAILSLFVN------VAGSSNPFLSRLLNRDTITRISYKNDAYFLQDLSLELLFPESFGFITYQGSLSTPPCSE : 248 CAH11_Hsap : NFSAASRGPNGLAILSLFVN------VASTSNPFLSRLLNRDTITRISYKNDAYFLQDLSLELLFPESFGFITYQGSLSTPPCSE : 248 CAH11_Ptro : NFSAASRGPNGLAILSLFVN------VASTSNPFLSRLLNRDTITRISYKNDAYFLQDLSLELLFPESFGFITYQGSLSTPPCSE : 248 CAH11_Pabe : NFSAASRGPNGLAILSLFVN------VASTSNPFLSRLLNRDTITRISYKNDAYFLQDLSLELLFPESFGFITYQGSLSTPPCSE : 248 CAH11_Ppyg : NFSAASRGPNGLAILSLFVN------VASTSNPFLSRLLNRDTITRISYKNDAYFLQDLSLELLFPESFGFITYQGSLSTPPCSE : 248 CAH11_Mmul : NFSAASRSPNGLAILSLFVN------VAGSSNPFLSRLLNRDTITRISYKNDAYFLQDLSLELLFPESFGFITYQGSLSTPPCSE : 248 CAH11_Cjac : NFSAASRGPNGLAILSLFVN------VAGSSNPFLSRLLNRDTITRISYKNDAYFLQDLSLELLFPESFGFITYQGSLSTPPCSE : 249 CAH11_Ttru : NLSAASRGPNGLAILSLFVN------VAGSSNPFLSRLLNRDTITRISYKNDAYFLQDLSLELLFPESFGFITYQGSLSTPPCSE : 248 CAH11_Oari : NLSAATRGPNGLAILSLFVN------VAGSSNPFLSRLLNRDTITRISYKNDAYFLQDLSLELLFPESFGFITYQGSLSTPPCSE : 248 CAH11_Cpor : NLSTASRGPNGLAILSLFVN------VAGSSNPFLSRLLNRDTITRISYKNDAYFLQDLSLELLFPESFGFITYQGSLSTPPCSE : 226 CAH11_Mdom : NLSAASRGPNGLAVLALFVNQPSPTQVAANSNPFLSRLLNRDTITRISYKNDAYFVQDLSLEQLFPESFGFITYQGSMSTPPCSE : 254 CAH11_Acar : NISEASRNPNGLAVISIFAN------IGPSPNPFLTRLLNRETITRISYKNDAYLLHDLSLEDLYPETFGFITYQGSMTTPPCYE : 217 CAH11_Xtro : NVTEASRNPNGLAVISLFLN------VADNSNSFLNRMLNRETITRISFRNEATYIQDLSIEDLYPDTFGFLTYQGSMTIPPCYE : 235 260 * 280 * 300 * 320 * CAH11_Rnor : TVTWILIDRALNITSLQ-MHSLRLLSQNPPSQIFQSLSGNGRPLQPLAHRALRGNRDPRHPERRCRGPNYRLHVDGGPHGR--- : 328 CAH11_Mmus : TVTWILIDRALNITSLQ-MHSLRLLSQNPPSQIFQSLSGNGRPLQPLAHRALRGNRDPRHPERRCRGPNYRLHVDGGPHGR--- : 328 CAH11_Fcat : TVTWILIDRALNITSLQ------: 265 CAH11_Ecab : TVTWILIDRALNITSLQVILCLHHLSALPP------: 278 CAH11_Sscr : TVTWILIDRALNITSLQ-MHSLRLLSQNPPSQIFQSLSGNGRPLQPLAHRALRGNRDPRHPERRCRGPNYRLHVDGAPHGR--- : 331 CAH11_Btau : TVTWILIDRALNITSLQ-MHSLRLLSQNPPSQIFQSLSGNGRPLQPLAHRALRGNRDPRHPERRCRGPNYRLHVDGAPHGR--- : 328 CAH11_Cfam : TVTWILIDRALNITSLQ-MHSLRLLSQNPPSQIFQSLSGNGRPLQPLAHRALRGNRDPRHPERRCRGPNYRLHVDGAPHGR--- : 328 CAH11_Hsap : TVTWILIDRALNITSLQ-MHSLRLLSQNPPSQIFQSLSGNSRPLQPLAHRALRGNRDPRHPERRCRGPNYRLHVDGVPHGR--- : 328 CAH11_Ptro : TVTWILIDRALNITSLQ-MHSLRLLSQNPPSQIFQSLSGNSRPLQPLAHRALRGNRDPRHPERRCRGPNYRLHVDGVPHGR--- : 328 CAH11_Pabe : TVTWILIDRALNITSLQ-MHSLRLLSQNPPSQIFQSLSGNSRPLQPLAHRALRGNRDPRHPERRCRGPNYRLHVDGAPHGR--- : 328 CAH11_Ppyg : TVTWILIDRALNITSLQ-MHSLRLLSQNPPSQIFQSLSGNSRPLQPLAHRALRGNRDPRHPERRCRGPNYRLHVDGAPHGR--- : 328 CAH11_Mmul : TVTWILIDRALNITSLQ-MHSLRLLSQNPPSQIFQSLSGNGRPLQPLAHRALRGNRDPRHPERRCRGPNYRLHVDGAPHGR--- : 328 CAH11_Cjac : TVTWILIDRALNITSLQ-MHSLRLLSQNPPSQIFQSLSGNGRPLQPLAHRALRGNRDPRHPERRCRGPNYRLH------: 321 CAH11_Ttru : TVTWILIDRALNITSLQ-MHSLRLLSQNPPSQIFQSLSGNGRPLQPLTHRALRGNRDPRHPERRCRGPNYRLH------: 320 CAH11_Oari : TVTWILIDRALNITSLQ-MHSLRLLSQNPPSQIFQSLSGNGRPLQPLAHRALRGNRDPRHPERRCRGPNYRLHVDDVPHGL--- : 328 CAH11_Cpor : TVTWILIDRPLNITSLQ-MHSLRLLSQNPPSQIFQSLSGNGRPLQPLAHRALRGNRDPRHPERRCRGPNYRLH------: 298 CAH11_Mdom : TVTWILIDRALNVTSLQ-MHSLRLLSQNPPSQIFQSLSGNGRPLQPLAHRALRGNRDSRHPERRCRGPNYRLHG--MPWSL--- : 332 CAH11_Acar : TATWILIDRPINITSVQ-IHSLRLLSQNLPSQIFRSMSDNSRPLQPLLHRSLRGNRDPRRPTRRCKGGNFRLH------: 289 CAH11_Xtro : TVTWIVIDRPLNITSMQ-MHSLRLLSQNQPSQIFQSMSDNFRPVQPLFHRGLRGNLDFRKPGRKCKGANYKLHIDRPTSSKNAN : 318

Figure 3 Multiple sequence alignment of CARP XI sequences. Comparison of 19 CARP XI sequences by multiple sequence alignment. Short names (first letter of the genus and first three letters of the species) are provided on the left side and residue numbers are provided on the right. Details of the sequences and full species names are provided in Table 1. Aspatwar et al. BMC Molecular Biology 2010, 11:25 Page 8 of 19 http://www.biomedcentral.com/1471-2199/11/25

lizards, and two smaller groups containing sequences from fishes (Figure 4). The CARP XI group contains a major branch, consisting of mammalian sequences, while the sequences from lizard and frog form an outgroup. The tree indicates that there has been an independent duplication of the ancestral vertebrate CA10 gene in the fish lineage, whereas we conclude that the CA11 gene has emerged from another gene duplication after the separation of the fish and tetrapod lineages.

Quantitative analysis of Car 8, Car10, Car11 mRNA expression in mouse tissues We studied the expression of all three CARP mRNAs in 20 different mouse tissues. The expression patterns of each mRNA are shown in Figures 5 through 7. As predicted based on previous studies [15], Car8 mRNA expression was found to be highest in the cerebellum, and high levels were also detected in the liver and the lung (Figure 5). Low expression was observed in the stomach, duodenum, ileum, jejunum, colon, spleen, kidney, heart, frontal cortex, parietal cortex, midbrain, and eye, while extremely low expression was observed in the ovary, skeletal muscle, and testis. The expression profile for Car10 mRNA is presented in Figure 6. The Car10 mRNA levels were high in the cerebellum, frontal cortex, and parietal cortex, low in the midbrain, and extremely low in the eye. The distribution of Car11 mRNA expression was broad, and the highest signals were observed in the cerebellum and cerebral cortex (Figure 7) while the midbrain showed moderate levels of expression. In addition to the brain, low expression was detected in the colon, kidney, ovary, heart, and lung. Moreover, barely detectable expression was observed in the liver, stomach, duodenum, ileum, jejunum, spleen, and eye.

Distribution of CARP VIII, X, and XI proteins in mouse tissues We studied the expression of CARP VIII, X, and XI proteins in mouse tissues using immunohistochemistry as shown in Figures 8 through 11 and in Table 2. CARP VIII was expressed in most of the tissues analyzed, indicating a wider distribution profile compared to CARP X and CARP XI expression. Strong expression for CARP VIII was observed in the cerebellum and cerebrum while weaker expression was observed in several other tissues including the liver, pancreatic Langerhans islets, submandibular gland, stomach, colon, kidney, and lung (Figures 8 and 9). In the cerebellum, the highest staining intensity was present in the Purkinje cells and a slightly lower staining intensity was associated with the molecular layer. The cerebrum showed an intense and punctate staining Figure 4 Evolutionary relationships of CARP sequences. The phylogenetic tree of 84 CARP sequences used in the study was inferred using pattern, indicating the strongest expression in the axons the Neighbor-Joining method. The percentage of replicate trees in and dendrites. In the kidney, very weak positive staining which the associated sequences clustered together in the bootstrap test is shown above the branches. Aspatwar et al. BMC Molecular Biology 2010, 11:25 Page 9 of 19 http://www.biomedcentral.com/1471-2199/11/25

Figure 5 Expression pattern of Car8 mRNA in different murine tissues. was observed in a few epithelial cells of the renal tubules. Discussion The liver showed moderate immunostaining in the hepa- Previous bioinformatic investigations have described tocytes. In the lung, staining was observed in both the individual CARP sequences only for human, mouse, and respiratory epithelium and the rounded alveolar cells, other mammals [10,11,25,26]. Using a variety of bioinfor- most likely representing the type II pneumocytes. The matic tools we identified 84 full-length sequences from submandibular gland showed strong immunoreactions in genome and sequence databases, including 22 coming both the acinar and ductal epithelial cells. The gastric and from novel or improved gene models and combining colonic glands were also positively stained for CARP VIII. mRNA data to genome data. Of these 22 sequences, 8 are A significant amount of CARP X expression was novel and previously unannotated, and 14 comprise observed only in the lung where the staining was local- extended and/or partially corrected sequences. ized to the respiratory epithelium. In addition, positive Sequences encoding CARP VIII and CARP X were iden- signals were occasionally detected in the cerebral capil- tified in all available vertebrate genomes, even if many laries and the stomach (Figures 8 and 10 and Table 2). sequences were present only as gene fragments. In con- Barely detectable staining was occasionally observed in trast, CARP XI was found only in mammals, frogs (X. the heart muscle cells. tropicalis), and lizards (A. carolinensis). Our results indi- CARP XI showed a broader expression profile than cate that the CA11 gene emerged through a process of CARP X. Its overall distribution was fairly similar to that gene duplication from CA10 after the divergence of the for CARP VIII, although the staining intensity was clearly fish and tetrapod lineages. In addition, we failed to iden- less intense. Positive signal for CARP XI was observed in tify any CARP XI sequences from birds, but since there the cerebellum, cerebrum, liver, stomach, small intestine, are only two genomes available, the CA11 gene may have colon, kidney, and testis (Figures 8 and 11 and Table 2). In been either missed as a consequence of the incomplete the cerebellum, the most prominent signal was located in genome coverage or alternatively the gene may have been the Purkinje cells. lost in the bird lineage. Aspatwar et al. BMC Molecular Biology 2010, 11:25 Page 10 of 19 http://www.biomedcentral.com/1471-2199/11/25

Figure 6 Expression profile of Car10 mRNA in murine tissues.

The fish CARP X-like sequences form two distinct sub- Earlier studies reported the expression of human or groups, both of which are more closely related to CARP X mouse CARP mRNAs and their corresponding proteins than CARP XI. This indicates that these sequences have using immunohistochemistry, western blot analysis, arisen by gene duplication from the CA10 gene in the fish northern blot assays, dot blots, and RT-PCR mostly in the lineage. We suggest names CA10a and CA10b for these brain and in a few cases in other tissues, often with genes and CARP Xa and CARP Xb for the proteins to dis- slightly conflicting results [12,13,16,22,27-30]. However, a tinguish them from CA11/CARP XI, which is specific to comprehensive distribution of all CARPs and their corre- tetrapods. sponding mRNAs has yet to be completely elucidated. In Sequences found in three deuterostomes (B. floridae, T. fact, the present study revealed for the first time the dis- adhaerens, and S. purpuratus) that were similar to CARP tribution of all CARP proteins and their mRNAs in a wide VIII, with sequence similarities between 40% and 45% variety of adult mouse tissues using immunohistochemis- when compared to vertebrate CARP VIII sequences, were try and real-time qPCR. novel and unexpected discoveries. No CARP VIII The expression of Car8 mRNA has been previously orthologs were likewise discovered in protostomes. studied in some other tissues besides from the brain According to these findings, the origin of CARP VIII using RT-PCR and dot blot analysis. Multiple transcripts would have occurred sometime after the separation of the were reported to be expressed in the brain, lung, and Protostomia and Deuterostomia lineages. In contrast, liver, and a single transcript was found in the heart, skele- CARP X-like sequences are widely found in invertebrates, tal muscle, and kidney, though to a lesser extent [11]. In including all available insect and nematode genomes. the present study, we observed high expression of Car8 Further analysis of the invertebrate CARP X homologs mRNA in the cerebellum, liver, and lung. Low expression will be presented elsewhere. of Car8 mRNA was observed in all other tissues except for the pancreas and uterus. In brief, the expression pat- Aspatwar et al. BMC Molecular Biology 2010, 11:25 Page 11 of 19 http://www.biomedcentral.com/1471-2199/11/25

Figure 7 Distribution of Car11 mRNA in murine tissues. tern we observed by RT-qPCR was similar to the earlier RT-PCR analyses have previously shown the expression results [11]. The immunohistochemical findings show of Car10 mRNA in the human brain, testis, salivary high levels of CARP VIII expression in the cerebellum glands, and kidney, while lower expression levels were and cerebrum. In previous studies, immunohistochemi- reported in the pancreas, liver, and testis [11]. Using cal staining, northern blot studies, and RT-PCR analyses northern blot analysis, the expression was observed in the have shown abundant expression of CARP VIII in the kidney and the brain [11]. Incidentally, there have been human and mouse brain, and immunohistochemical only a few previous reports in the literature regarding the studies have further defined its expression especially in expression of the CARP X protein in the human and the cerebellar Purkinje cells [12,13,16,22,27-30], all of mouse brain. The expression was shown to be weak in the which are in agreement with the results of our study. cerebellar Purkinje cells [14,16]. Herein we report strong Lower CARP VIII expression has been reported in other positive signal intensities only in the cerebellum followed murine tissues including the lung, liver, and stomach by the parietal cortex and the frontal cortex, low expres- using western blot analysis [30]. Our study confirmed the sion in the midbrain, and extremely low expression in the expression using immunohistochemical staining in these eye. Using immunohistochemical studies, we observed organs. High level expression of Car8 mRNA and CARP clearly localized signal only in the respiratory epithelium VIII protein in the brain and in a wide variety of other tis- of the lung, and weak signals in the stomach and cerebral sues suggests important roles for this protein in normal capillaries. Our real-time qPCR results do not agree with physiology. Indeed the role of CARP VIII is evident from previous results on human tissues (except for the brain) the recently published reports showing ataxia and gait nor do they agree with the present immunohistochemical disorders in both mice and humans due to mutations in findings. The discrepancies between immununohis- the CA8 gene [22,23]. tochemistry and RT-qPCR may be due to (i) a low amount of mRNA that is translated into protein; (ii) rapid Aspatwar et al. BMC Molecular Biology 2010, 11:25 Page 12 of 19 http://www.biomedcentral.com/1471-2199/11/25

Figure 8 Immunohistochemical staining of CARP VIII, CARP X, and CARP XI proteins in mouse cerebellum (A, C, E) and cerebrum (B, D, F). Arrows indicate the location of the Purkinje cells, which are strongly positive for CARP VIII and moderately positive for CARP XI. The molecular layer (m) of the cerebellum is also intensely labeled with CARP VIII, whereas the granular cell layer is negative (g). Panel B shows strong punctuate immunostaining for CARP VIII in the cerebrum. The arrowheads in panel D indicate CARP X-positive microcapillaries in the cerebrum. Immunostaining re- actions for CARP XI remained very weak in the cerebrum (F). Original magnifications are at × 20. degradation of the protein; (iii) the low signal from storage of slides; (iv) differences in the species or strains immunochemical staining, which could be due to the loss used. Further immunohistochemical studies using new of antigenicity in some tissues during the processing and antibodies along with analysis of mRNA transcripts will Aspatwar et al. BMC Molecular Biology 2010, 11:25 Page 13 of 19 http://www.biomedcentral.com/1471-2199/11/25

Figure 9 Immunohistochemical staining of CARP VIII proteins in mouse tissues. CARP VIII was observed moderately in the liver hepatocytes (A), ducts (*) and acini (arrows) of the submandibular gland (C), gastric glands (arrows in D), respiratory epithelium (arrowhead), and rounded alveolar cells (arrows) of the lung (G). Extremely low expression was observed in the pancreatic Langerhans islets (*in B), colonic glands (arrows in E), and occasionally in the tubule cells of the kidney (arrow in F indicates positive macula densa cells). No staining was present in the skeletal muscle (H) and testis (I). Original magnifications are at × 20. Aspatwar et al. BMC Molecular Biology 2010, 11:25 Page 14 of 19 http://www.biomedcentral.com/1471-2199/11/25

Table 2: Expression profile of CARP VIII, X, and XI in mouse tissues using immunohistochemistry

Tissue CARP VIII CARP X CARP XI

Cerebellum +++ --- ++- Cerebrum +++ ++- +-- Liver ++- --- +-- Pancreas +------Submandibular gland ++------Stomach ++- +-- +-- Colon +-- --- +-- Kidney +-- ++- +-- Lung ++------Skeletal muscle ------Testis --- NA +- Ovary NA -- NA Heart NA +-- NA Epididymis NA ------Small intestine ------+-- +++ Strong, ++Moderate, +-Weak, ---No signal, and NA-Not analyzed be important for understanding the discrepancies the lung, skeletal muscle, kidney, pancreas, and liver [10]. observed. In our study, low levels of Car11 mRNA were observed in In a previous paper, Okamoto et al. [11] reported that the colon, kidney, ovary, heart, and lung. The presence of the Car10 sequence contains seven CCG repeats in the CARP XI in several regions of the brain suggests an 5'-untranslated region followed by two CCG repeats important, yet undefined role for CARP XI in the central located 16 bp downstream from the aforementioned nervous system. repeats. These repeats have been associated with various neurological disorders [31]. The presence of the CCG Conclusions repeats in the Car10 gene makes it a potential candidate The present investigation describes a comprehensive bio- gene that might contribute to the development of neuro- informatic study of CARP gene sequences and also eluci- degenerative disorders. Therefore, it will be of interest to dates the distribution of three CARPs in mouse tissues explore the expansion mutations of Car10 gene in using real-time qPCR and immunohistochemistry. We patients with neurological symptoms [8]. have observed a very high conservation of all the three Our study revealed widespread expression of CARP XI CARP sequences across the species. In the cases of CARP in most of the tissues studied by using both immunohis- VIII and CARP X, we found unusually high similarity tochemistry and real-time qPCR. Compared to CARP between vertebrate and invertebrate sequences. Based on VIII, the intensity of CARP XI immunostaining was our results, the duplication history of the CA10 gene has clearly weaker. In a previous study, immunochemical followed different paths in the fish and tetrapod lineages. staining of the human brain indicated that the signal for Our results contribute to a deeper understanding of CARP XI was lower compared to CARP VIII but higher CARP evolution across species. than CARP X [16]. Thus, our findings were in agreement The expression of CARP VIII was found to be wide- with these aforementioned results. The present real-time spread in the tissues analyzed, and the highest mRNA sig- qPCR analysis, surprisingly, showed very high expression nals were detected in the cerebellum, lung, and liver. Both of the Car11 mRNA in all of the brain segments analyzed, CARP X and XI showed the strongest mRNA expression especially in the frontal cortex followed by the parietal in the nervous tissues. The distribution patterns suggest cortex, cerebellum, and midbrain. These results were in that CARPs may contribute to the development of the agreement with an earlier report showing Car11 mRNA nervous system, motor coordination functions, and yet expression in all parts of the human brain [10]. The same unknown physiological roles in other tissues. It will be of study demonstrated Car11 mRNA expression in the kid- interest to determine the specific function of all CARPs ney, liver, and salivary glands, and low expression levels in by producing and analyzing suitable single, double, and Aspatwar et al. BMC Molecular Biology 2010, 11:25 Page 15 of 19 http://www.biomedcentral.com/1471-2199/11/25

Figure 10 Immunohistochemical staining of CARP X protein in mouse tissues. Moderate CARP X expression was observed in the respiratory epithelium (arrowhead) of the lung (G). Moderate expression was also present in the gastric glands (arrows in panel D). The heart muscle cells occasionally showed extremely weak signals (I). The other tissues including the liver (A), pancreas (B), submandibular gland (C), colon (E), small intestine (F), skeletal muscle (H) remained negative. Original magnifications are at × 20. Aspatwar et al. BMC Molecular Biology 2010, 11:25 Page 16 of 19 http://www.biomedcentral.com/1471-2199/11/25

Figure 11 Immunohistochemical staining of CARP XI protein in mouse tissues. Weak immunoreactions for CARP XI were observed in the crypts of Lieberkuhn (arrows in D), gastric glands (arrows in C), and renal tubule cells (F). Extremely weak signals can also be observed in the liver (A), colon (E), and testis (I). The pancreas (B), lung (G), and skeletal muscle (H) were all negative. Original magnifications are at × 20. Aspatwar et al. BMC Molecular Biology 2010, 11:25 Page 17 of 19 http://www.biomedcentral.com/1471-2199/11/25

triple knockout animal models and also by screening neu- methanol + 3% H2O2 for 5 min; (ii) rinsing with 1 × Tris- rological patients for trinucleotide repeats or other muta- buffered saline (TBS), pH 8.0, containing 0.05% Tween; tions in CARP genes. (iii) blocking with Rodent Block M™(Biocare Medical, Concord, CA) for 30 min; (iv) incubating with primary Methods rabbit anti-human CARP VIII, CARP X, and CARP XI Sequence retrieval antibodies (Santa Cruz Biotechnology, Inc. Bergheimer The available CARP protein sequences were obtained Heidelberg, Germany) raised against amino acids 1 to 100 from Ensembl [32], UniProt [33], and RefSeq [34] and (CARP VIII), 1 to 50 (CARP X) and 279 to 328 (CARP XI) further sequences were searched using BLAST from diluted 1:350 in 1% bovine serum albumin (BSA) in phos- NCBI protein databases [35] and via BLAT searches from phate-buffered saline (PBS) for 1 hour at room tempera- complete genomes [36] using human and mouse CARPs ture and rinsing with TBS containing 0.05% Tween. as initial query sequences, and zebrafish, lancelet and sea Notably, rabbit IgG was used as a control instead of the urchin CARPs later as they were discovered and con- primary antibodies; (v) incubation with Rabbit HRP- firmed. Duplicate sequences were rejected, after which Polymer + XM Factor™(Biocare Medical, Concord, CA) the remaining sequences were taken through iterated (2.5 ml HRP-Polymer + 1 to 2 drops XM Factor) for 30 cycles of multiple sequence alignment [37], evaluation, min prior to rinsing with 1 × TBS containing 0.05% and revision. For revision, sequences with poorly match- Tween; (vi) treatment with 1 × 3,3'-diaminobenzidine tet- ing or missing regions were subjected to gene model gen- rahydrochloride (DAB) solution for 5 min prior to rinsing eration with GeneWise [38] taking the genomic with distilled water; (vii) counterstaining of the slides sequences from the UCSC Genome Browser [36]. EST with Mayer's Hematoxylin for 1 to 3 sec and rinsing and mRNA sequence data were used to confirm gene under tap water for 10 min. After dehydration, the slides models, to bridge gaps or fill ends in the genomic were mounted with Entellan Neu™ (Merck; Darmstadt, sequences, and to discover and assemble CARPs from Germany), examined and photographed using Nikon less than genome-wide sequenced organisms. Finally, Microphot microscope (Nikon Microphot-FXA, Japan). incomplete sequences were rejected, with the exception All of the procedures were carried out at room tempera- of marginally shortened ends, which were allowed. ture.

Multiple sequence alignment RNA Extraction Individual multiple sequence alignments (MSAs) were Specimens from 20 tissue samples were collected from calculated for CARP VIII, CARP X, and CARP XI using six normal NMRI mice. The tissue samples used for ClustalW [39] and visualized using GeneDoc software mRNA isolation were stabilized in RNAlater (Ambion, [40]. Furthermore, all of the 84 CARP protein sequences Austin, TX, USA) immediately after collection, and the were aligned together for the phylogenetic tree. total RNA was isolated from 30 mg of tissue samples using the RNeasy Mini kit (Qiagen, Hilden, Germany) by Phylogenetic analysis following the manufacturer's instructions. The concen- The phylogenetic tree of all CARP protein sequences was tration and purity of RNA was determined using a spec- constructed from the MSA of 84 sequences using the trophotometric method at 260 and 280 nm. MEGA software, version 4.1 [41]. Evolutionary relationships were inferred using the Neighbor-Joining method. Quantitative real-time PCR A bootstrap test was performed using 1000 replicates and Reverse transcriptase PCR was performed using 50 μg of evolutionary distances were computed using the Poisson total RNA to synthesize the first strand of cDNA using correction method with the complete deletion option. First Strand cDNA Synthesis kits (High-Capacity cDNA Reverse Transcription Kits, Applied Biosystems, Foster Immunohistochemistry City, CA) with random primers and MultiScribe™ Reverse The tissue specimens from the normal mice were fixed in Transcriptase according to the protocol recommended by 4% neutral-buffered formaldehyde at +4°C for 8 to 27 the manufacturer. days. The samples were then dehydrated in an alcohol Real-time qPCR primers were designed based on the series, treated with xylene, embedded in paraffin wax, complete cDNA sequences deposited in the GenBank and 4 μm sections were cut and placed on Superfrost (RefSeq accession numbers: Car8 [NCBI: NM_007592], microscope slides. After removal of the paraffin with Car10 [NCBI: NM_028296], and Car11 [NCBI: xylene, the rehydrated sections were boiled in sodium cit- NM_009800]). Primer sequences for Car8 included the rate (0.01 M, pH 6.0) for 20 min and cooled down. The Forward primer (5'-3') cgggattactgggtctatgaagg and the sections were immunohistochemically stained according Reverse primer (5'-3') ggctgggtaggtcggaaattgtc, while the to the following procedure: (i) incubating the sample in primer sequences for Car10 included the Forward primer Aspatwar et al. BMC Molecular Biology 2010, 11:25 Page 18 of 19 http://www.biomedcentral.com/1471-2199/11/25

(5'-3') gagagcaagagcccagaactc and the Reverse primer (5'- 6. Bertucci A, Innocenti A, Zoccola D, Scozzafava A, Tambutte S, Supuran CT: Carbonic anhydrase inhibitors. Inhibition studies of a coral secretory 3') ctcaccagtggcagaaatggc. In addition, the Car11 primer isoform by sulfonamides. Bioorg Med Chem 2009, 17(14):5054-5058. sequences were (5'-3') gccggctctgaacaccagatc (for the For- 7. Tashian REH-ED, Carter ND, Bergenhem NCH, (ed): Carbonic anhydrase ward primer) and (5'-3') gaggaggcgactgaggaatgg (for the (CA)-related proteins (CA-RPs), and trans-membrane proteins with CA or CA RP membrane proteins with CA or CA RP domains. Basel: New Reverse primer). Horizons, Birkhauser; 2000. Real-time qPCR was performed using the SYBR Green 8. Nishimori I: Acatalytic CAs: Carbonic anhydrase-related proteins. In PCR Master Mix Kit in an ABI PRISM 7000 Detection Carbonic anhydrases: Its inhibitors and activators. CRC enzyme inhibitor series Edited by: Supuran CT, Scozzfava A, Conway J. CRC press; 2004:25-43. System™ according to the manufacturer's instructions 9. Barnea G, Silvennoinen O, Shaanan B, Honegger AM, Canoll PD, (Applied Biosystems). The PCR conditions consisted of D'Eustachio P, Morse B, Levy JB, Laforgia S, Huebner K, et al.: Identification an initial denaturation step at 95°C for 10 min followed by of a carbonic anhydrase-like domain in the extracellular region of RPTP gamma defines a new subfamily of receptor tyrosine phosphatases. 40 cycles at 95°C for 15 sec (denaturation) and 60°C for 1 Mol Cell Biol 1993, 13(3):1497-1506. min (elongation). The data were analyzed using the ABI 10. Fujikawa-Adachi K, Nishimori I, Taguchi T, Yuri K, Onishi S: cDNA PRISM 7000 SDS™ software (Applied Biosystems). Every sequence, mRNA expression, and chromosomal localization of human carbonic anhydrase-related protein, CA-RP XI. Biochim Biophys Acta PCR was performed in a total reaction volume of 15 μl 1999, 1431(2):518-524. containing 2 μl of first strand cDNA (20 ng cDNA), 1 × 11. Okamoto N, Fujikawa-Adachi K, Nishimori I, Taniuchi K, Onishi S: cDNA Power SYBR green PCR Master Mix™ (Applied Biosys- sequence of human carbonic anhydrase-related protein, CA-RP X: mRNA expressions of CA-RP X and XI in human brain. Biochim Biophys tems, Foster City, CA 94404, USA), and 0.5 μM of each Acta 2001, 1518(3):311-316. primer. The final results, expressed as the N-fold relative 12. Taniuchi K, Nishimori I, Takeuchi T, Fujikawa-Adachi K, Ohtsuki Y, Onishi S: difference (ratio) in gene expression between the studied Developmental expression of carbonic anhydrase-related proteins VIII, X, and XI in the human brain. Neuroscience 2002, 112(1):93-99. samples, were calculated according to the Pfaffl's equa- 13. Akisawa Y, Nishimori I, Taniuchi K, Okamoto N, Takeuchi T, Sonobe H, tion with appropriate modification [42]. Ohtsuki Y, Onishi S: Expression of carbonic anhydrase-related protein CA-RP VIII in non-small cell lung cancer. Virchows Arch 2003, 442(1):66-70. Additional material 14. Nishimori I, Takeuchi H, Morimoto K, Taniuchi K, Okamoto N, Onishi S, Ohtsuki Y: Expression of carbonic anhydrase-related protein VIII, X and Additional file 1 Multiple sequence alignment of CARP VIII, X, and XI XI in the enteric autonomic nervous system. Biomed Res 2003, sequences. Multiple sequence alignment of the 84 CARP protein 14(1):69-73. sequences analyzed in the study which was used for the construction of 15. Taniuchi K, Nishimori I, Takeuchi T, Ohtsuki Y, Onishi S: cDNA cloning and the phylogenetic tree. developmental expression of murine carbonic anhydrase-related proteins VIII, X, and XI. Brain Res Mol Brain Res 2002, 109(1-2):207-215. Authors' contributions 16. K Taniuchi IN, Takeuchi T, Fujikawa-Adachi K, Ohtsuki Y, Onishi S: All authors participated in the design of the study. AA is the corresponding Developmental expression of carbonic anhydrase-related proteins VIII, author who performed the experimental work and drafted the first version of X, and XI in the human brain. Neuroscience 2002, 112:93-99. the manuscript. AA and MT jointly performed the bioinformatic analyses, 17. Miyaji E, Nishimori I, Taniuchi K, Takeuchi T, Ohtsuki Y, Onishi S: under supervision of MT. In addition, AA and SP performed essential micro- Overexpression of carbonic anhydrase-related protein VIII in human scopic analyses. All authors contributed to the writing of the document and colorectal cancer. J Pathol 2003, 201(1):37-45. approved the final version of the manuscript. 18. Morimoto K, Nishimori I, Takeuchi T, Kohsaki T, Okamoto N, Taguchi T, Yunoki S, Watanabe R, Ohtsuki Y, Onishi S: Overexpression of carbonic Acknowledgements anhydrase-related protein XI promotes proliferation and invasion of We thank Marianne Kuuslahti and Aulikki Lehmus for their skilful technical gastrointestinal stromal tumors. Virchows Arch 2005, 447(1):66-73. 19. Lu SH, Takeuchi T, Fujita J, Ishida T, Akisawa Y, Nishimori I, Kohsaki T, Onishi assistance with the experiments and Csaba Ortutay for helpful discussions. This S, Sonobe H, Ohtsuki Y: Effect of carbonic anhydrase-related protein VIII work was supported by the Competitive Research Funding of the Tampere expression on lung adenocarcinoma cell growth. Lung Cancer 2004, University Hospital (Grant 9L071) and a grant from Academy of Finland. 44(3):273-280. 20. Ishihara T, Takeuchi T, Nishimori I, Adachi Y, Minakuchi T, Fujita J, Sonobe Author Details H, Ohtsuki Y, Onishi S: Carbonic anhydrase-related protein VIII increases 1 Bioinformatics Group, Institute of Medical Technology, 33014 University of invasiveness of non-small cell lung adenocarcinoma. Virchows Arch 2 Tampere, Tampere, Finland, Institute of Medical Technology and School of 2006, 448(6):830-837. 3 Medicine, University of Tampere, 33520 Tampere, Finland and Centre for 21. Nishikata M, Nishimori I, Taniuchi K, Takeuchi T, Minakuchi T, Kohsaki T, Laboratory Medicine, Tampere University Hospital, 33520 Tampere, Finland Adachi Y, Ohtsuki Y, Onishi S: Carbonic anhydrase-related protein VIII promotes colon cancer cell growth. Mol Carcinog 2007, 46(3):208-214. Received: 24 November 2009 Accepted: 31 March 2010 22. Jiao Y, Yan J, Zhao Y, Donahue LR, Beamer WG, Li X, Roe BA, Ledoux MS, Published: 31 March 2010 This©BMC 2010 isarticleMolecular an Aspatwar Open is available Biology Access et al; from:2010,articlelicensee http11 distributed :25BioMed://www.biomedcentral.com/1471-2199/11/25 Central under Ltd.the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Gu W: Carbonic anhydrase-related protein VIII deficiency is associated with a distinctive lifelong gait disorder in waddles mice. Genetics 2005, References 171(3):1239-1246. 1. Henry RP: Multiple roles of carbonic anhydrase in cellular transport and 23. Turkmen S, Guo G, Garshasbi M, Hoffmann K, Alshalah AJ, Mischung C, metabolism. Annu Rev Physiol 1996, 58:523-538. Kuss A, Humphrey N, Mundlos S, Robinson PN: CA8 mutations cause a 2. Supuran CT: Carbonic anhydrases: novel therapeutic applications for novel syndrome characterized by ataxia and mild mental retardation inhibitors and activators. Nat Rev Drug Discov 2008, 7(2):168-181. with predisposition to quadrupedal gait. PLoS Genet 2009, 3. Sly WS, Hu PY: Human carbonic anhydrases and carbonic anhydrase 5(5):e1000487. deficiencies. Annu Rev Biochem 1995, 64:375-401. 24. Picaud SS, Muniz JR, Kramm A, Pilka ES, Kochan G, Oppermann U, Yue 4. Tashian RE: The carbonic anhydrases: widening perspectives on their WW: Crystal structure of human carbonic anhydrase-related protein evolution, expression and function. Bioessays 1989, 10(6):186-192. VIII reveals the basis for catalytic silencing. Proteins 2009, 76(2):507-511. 5. Tashian RE: Genetics of the mammalian carbonic anhydrases. Adv Genet 1992, 30:321-356. Aspatwar et al. BMC Molecular Biology 2010, 11:25 Page 19 of 19 http://www.biomedcentral.com/1471-2199/11/25

25. Kato K: Sequence of a novel carbonic anhydrase-related polypeptide and its exclusive presence in Purkinje cells. FEBS Lett 1990, 271(1- 2):137-140. 26. Bellingham J, Gregory-Evans K, Gregory-Evans CY: Sequence and tissue expression of a novel human carbonic anhydrase-related protein, CARP-2, mapping to chromosome 19q13.3. Biochem Biophys Res Commun 1998, 253(2):364-367. 27. Hirasawa M, Xu X, Trask RB, Maddatu TP, Johnson BA, Naggert JK, Nishina PM, Ikeda A: Carbonic anhydrase related protein 8 mutation results in aberrant synaptic morphology and excitatory synaptic function in the cerebellum. Mol Cell Neurosci 2007, 35(1):161-170. 28. Antal Nógrádi NJ, Richard Walker, Keith Caddy, Nick Carter, Christiane Kelly: Carbonic anhydrase II and carbonic anhydrase-related protein in the cerebellar cortex of normal and lurcher mice. Developmental Brain Research 1997, 98:91-101. 29. Lakkis MM, O'Shea KS, Tashian RE: Differential expression of the carbonic anhydrase genes for CA VII (Car7) and CA-RP VIII (Car8) in mouse brain. J Histochem Cytochem 1997, 45(5):657-662. 30. Junji Hirota HA, Kozo Hamada, Katsuhiko Mikoshiba: Carbonic anhydrase-related protein is a novel binding protein for inositol 1,4,5- trisphosphate receptor type 1. Biochem J 2003, 372:435-441. 31. Kleiderlein JJ, Nisson PE, Jessee J, Li WB, Becker KG, Derby ML, Ross CA, Margolis RL: CCG repeats in cDNAs from human brain. Hum Genet 1998, 103(6):666-673. 32. Ensembl genome database [http://www.ensembl.org/] 33. Universal Protein Resource [http://www.uniprot.org/] 34. Non-redundant database of DNA, RNA, and protein sequences [http:// www.ncbi.nlm.nih.gov/RefSeq/] 35. Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ: Basic local alignment search tool. J Mol Biol 1990, 215(3):403-410. 36. UCSC Genome Browser [http://genome.ucsc.edu/] 37. Larkin MA, Blackshields G, Brown NP, Chenna R, McGettigan PA, McWilliam H, Valentin F, Wallace IM, Wilm A, Lopez R, et al.: Clustal W and Clustal X version 2.0. Bioinformatics 2007, 23(21):2947-2948. 38. Birney E, Clamp M, Durbin R: GeneWise and Genomewise. Genome Res 2004, 14(5):988-995. 39. Align Sequences using Clustal W2 EBI [http://www.ebi.ac.uk/Tools/ clustalw2/] 40. Nicholas KB, Nicholas HB Jr, Deerfield DW II: GeneDoc: Analysis and Visualization of Genetic Variation. EMBNEWNEWS 1997, 4:14. 41. Tamura K, Dudley J, Nei M, Kumar S: MEGA4: Molecular Evolutionary Genetics Analysis (MEGA) software version 4.0. Mol Biol Evol 2007, 24(8):1596-1599. 42. Pfaffl MW: A new mathematical model for relative quantification in real- time RT-PCR. Nucleic Acids Res 2001, 29(9):e45.

doi: 10.1186/1471-2199-11-25 Cite this article as: Aspatwar et al., Phylogeny and expression of carbonic anhydrase-related proteins BMC Molecular Biology 2010, 11:25 Human Molecular Genetics, 2013, Vol. 22, No. 3 417–432 doi:10.1093/hmg/dds438 Advance Access published on October 18, 2012 Abnormal cerebellar development and ataxia in CARP VIII morphant zebraﬁsh

Ashok Aspatwar1,∗, Martti E.E. Tolvanen2, Eija Jokitalo3, Mataleena Parikka2, Csaba Ortutay2, Sanna-Kaisa E. Harjula2, Mika Ra¨met2, Mauno Vihinen2,4,5 and Seppo Parkkila1,4

1Institute of Biomedical Technology and School of Medicine, 2Institute of Biomedical Technology, University of Downloaded from Tampere and BioMediTech, Tampere 33014, Finland, 3Electron Microscopy Unit, Institute of Biotechnology, University of Helsinki, Helsinki 00014, Finland, 4Fimlab Laboratories, University of Tampere Hospital, Tampere 33014, Finland and 5Department of Experimental Medical Science, Lund University, Lund, Sweden

Received September 19, 2012; Revised September 19, 2012; Accepted October 11, 2012 http://hmg.oxfordjournals.org/

Congenital ataxia and mental retardation are mainly caused by variations in the genes that affect brain development. Recent reports have shown that mutations in the CA8 gene are associated with mental retardation and ataxia in humans and ataxia in mice. The gene product, carbonic anhydrase-related protein VIII (CARP VIII), is predominantly present in cerebellar Purkinje cells, where it interacts with the inositol 1,4,5-trisphosphate receptor type 1, a calcium channel. In this study, we investigated the effects of the loss of function of CARP VIII during embryonic development in zebrafish using antisense morpholino oligonucleotides at Tampere University Library. Department of Health Sciences on May 19, 2014 against the CA8 gene. Knockdown of CA8 in zebrafish larvae resulted in a curved body axis, pericardial edema and abnormal movement patterns. Histologic examination revealed gross morphologic defects in the cerebellar region and in the muscle. Electron microscopy studies showed increased neuronal cell death in developing larvae injected with CA8 antisense morpholinos. These data suggest a pivotal role for CARP VIII during embryonic development. Furthermore, suppression of CA8 expression leads to defects in motor and coordination functions, mimicking the ataxic human phenotype. This work reveals an evolutionarily conserved function of CARP VIII in brain development and introduces a novel zebrafish model in which to investigate the mechanisms of CARP VIII-related ataxia and mental retardation in humans.

INTRODUCTION The members of the mammalian a-carbonic anhydrase (a-CA) gene family are zinc-containing metalloenzymes that Neurodegenerative disorders are a complex group of diseases catalyze the reversible hydration of carbon dioxide (4–6). caused mainly by genetic factors leading to motor disturbance, There are typically 16 isoforms in a-CA gene family in cognitive loss and psychiatric manifestations. Several genes most mammals, of which 13 isozymes (CA I, II, III, IV, have reported involvement in the development of neurologic VA, VB, VI, VII, IX, XII, XIII, XIV and XV) are catalytically disorders in humans, mostly due to mutations. In many active. Three of the isoforms, namely, carbonic instances, the genetic causes may involve as yet unknown anhydrase-related proteins (CARPs) VIII, X and XI, represent genes. Recent studies in humans have shown that mutations catalytically inactive isoforms due to the lack of one or more in the CA8 gene lead to mental retardation, ataxia and, in of the three histidine residues required for CA catalytic activ- some instances, a quadrupedal gait (1,2). Similarly, a spontan- ity (7). eously occurring mutation in the Car8 gene in waddle (wdl) Previous studies (8), including our recent investigations (9), mice leads to ataxia and a lifelong gait disorder (3). These showed predominant expression of all the CARPs in the brain. studies demonstrate that novel genes with poorly characterized Expression analysis using both reverse transcriptase- functions may be involved in the development of neurodegen- quantitative PCR (RT-qPCR) and immunohistochemistry erative disorders in humans. showed that carbonic anhydrase-related protein VIII (CARP

∗To whom correspondence should be addressed. Tel: +358-503186251; Fax: +358-3-3641-482; Email: ashok.aspatwar@uta.ﬁ

VIII) is highly expressed in the Purkinje cells of the cerebel- human CARP VIII proteins (291 and 290 amino acids, respect- lum, whereas CARP X and XI show a more widespread distri- ively). The difference is mainly due to the absence of seven bution throughout the central nervous system (CNS). Although glutamic acid (E) residues at the N-terminus of zebrafish the wide distribution patterns suggest important roles for CARP VIII. CARPs in the CNS, their precise functions are still unclear. Sequencing of the CA8 cDNA revealed nucleotide varia- Phylogenetic analysis revealed that a highly conserved tions at three places, but the amino acid sequence deduced ortholog of human CARP VIII is present in zebrafish, with from the nucleotide sequence did not show any change in 79% protein sequence identity compared with human CARP the amino acid sequence, when compared with the sequence VIII (9,10). Expression analysis showed that the CA8 gene from the database (Supplementary Material, Figure S1). is expressed in both the zebrafish larvae and adult fish. This The high degree of homology between the CARP VIII prompted us to use zebrafish as a model organism to knock- sequences suggests a conserved and essential function. CARP Downloaded from down the CA8 gene to study the role of CARP VIII during em- VIII interacts with the inositol 1,4,5-trisphosphate receptor bryonic development. type 1 (ITPR1), an ion channel protein that regulates internal Many of the genes involved in neurodegenerative disorders Ca2+ ion release (14). A schematic of the primary structure play important roles in brain development during embryogen- of ITPR1 is shown in Supplementary Material, Figure S2. It

esis. Although the structure of the brain exhibits considerable contains major domains for binding calmodulin and inositol http://hmg.oxfordjournals.org/ differences among the vertebrate species, the molecular basis trisphosphate (IP3), a channel domain and a modulatory of brain development is relatively conserved (11). The zebra- domain. Deletion mutants have been used to map the binding ﬁsh has recently emerged as an attractive model organism for of CARP VIII to the region of amino acids 1387–1647 studying vertebrate development, as it uniquely combines the within the modulatory domain (14). The binding of CARP advantages of genetic tractability with biologic relevance VIII to ITPR1 is believed to reduce the sensitivity of ITPR1 (12,13). to inositol trisphosphate (IP3) and control the release of Ca2+ Thus far, there are neither studies on the role of CARP VIII ions from the internal stores of the endoplasmic reticulum during embryonic development in vertebrates, nor are there (15). Deletion mutagenesis experiments in a yeast two-hybrid

any targeted gene knockout or knockdown studies on CARP system showed that almost the entire CA domain of CARP at Tampere University Library. Department of Health Sciences on May 19, 2014 VIII to evaluate its function. Here, we investigate the role of VIII is required for ITPR1 binding. Only one of the truncated CARP VIII in the zebrafish model during embryogenesis. constructs, which was missing the first 44 residues, showed binding, and even then, the reaction was slower (14). To assess the functional association between ITPR1 and RESULTS CARP VIII throughout the vertebrate evolution, we performed two coevolution analyses of genes coding for CARP VIII and Bioinformatic analysis of zebrafish CARP VIII suggests ITPR1 in 31 species. In one analysis, the ITPR1 sequences an interaction with inositol 1,4,5-trisphosphate included the 6 fish orthologs for itpr1a of Danio rerio together receptor type 1 (ITPR1) with 25 tetrapod sequences. In another analysis, the six fish Similar to mice and humans, zebrafish have an ortholog of orthologs for itpr1b of D. rerio were used instead. All the CA8 gene, as reported previously (9). We obtained the ITPR1 sequences were restricted to the region of approximate- zebrafish CA8 transcript and protein sequences from ly 270 amino acids corresponding to the CARP VIII binding Ensembl (transcript ID: ENSDART00000057098, protein region in ITPR1 (14). Our results from the itpr1a orthologs ID: ENSDARP00000057097). The zebrafish transcript is show that the two protein sequences have several coevolving encoded by a 23.49 kb gene located on the forward strand of sites that are significant (chi-squared test, P , 0.001). chromosome 2. The gene contains 9 exons that code for 281 The analysis with the fish itpr1b orthologs also suggested amino acids, whereas the mouse and human transcripts are coevolution, but with a lower P-value (P , 0.05). This indi- encoded by genes of more than 90 kb in length, which are cates that the gene product of itpr1a is more likely to have located on the reverse strand of chromosomes 4 and 8, respect- retained the interaction with CARP VIII in fish. ively. A major portion of exon 1, a small part of exon 8, and When the results of the coevolution analyses are projected all of exon 9 are non-coding, similar to mouse and human on the 3D structure of human CARP VIII, we can observe a transcripts (Fig. 1A). The organization of coding exons in cluster of surface residues in which variations correlate strong- CA8 of zebrafish is the same as in all orthologs we have ly with variations in ITPR1 binding (Fig. 2B). Within residues studied. Figure 1B shows a representation of aligned protein 150–157, five residues show correlation coefficients of 0.95 or sequences, which highlights the fact that all exon boundaries higher. They are localized in a single loop region, and both in CA8 have been maintained in the same locations through known human disease-causing CA8 mutations are near this deuterostome evolution. region. The G162R mutation (1) is found in the end of this Comparisons of the deduced amino acid sequence of the loop. Any non-glycine residue in this position would cause zebrafish CARP VIII (Ensembl protein ID: ENSDARP a large change in the conformation and/or localization of 00000057097) with the human and mouse CARP VIII the loop. Regarding this location, the researchers who first sequences displayed a 79% overall amino acid identity, and reported this location actually depicted G193 instead of the amino acid identity within the CA domain (amino acids G162 in their figure (1). The mutation S100P (2) is in the 40–290 in the human sequence) was 84% (Fig. 2A). The adjacent loop (Fig. 2B). This mutation is likely to change deduced amino acid sequence of zebrafish CARP VIII is the conformation of the entire loop 100–109, which would slightly shorter (281 amino acids) than the mouse and also interfere with the loop 147–162. Human Molecular Genetics, 2013, Vol. 22, No. 3 419 Downloaded from http://hmg.oxfordjournals.org/ at Tampere University Library. Department of Health Sciences on May 19, 2014

Figure 1. Exon structures: (A) A comparison of the CA8 transcripts of human, mouse and zebrafish. A schematic representation of transcripts of human (ID: ENST00000317995), mouse (ID: ENSMUST00000066674) and zebrafish (ID: ENSDART00000057098). The exons are represented by vertical bars. The closed bars represent the coding exons, and the open bars are the non-coding exons. The introns are shown as horizontal bars. (B) The organization of coding exons in different organisms when compared with that of zebrafish, derived from a protein alignment. The gaps correspond to the alignment, and consecutive exons are shown in different shades. The exon boundaries have been retained in the same locations throughout the deuterostome evolution.

The coevolution results and the examination of mutation was expressed in almost all of the tissues analyzed sites lead us to suggest that the region that contains residues (Fig. 3B). The expression was high in the brain, heart, 150–157 interacts with ITPR1. The coevolving and possibly kidney, eye and skin. Low levels of expression were observed interacting residues in ITPR1 are 15 residues in the range in the male and female gonads, bowel, liver and gills, and very 1437–1630 (numbering by human ITPR1, isoform 1). low levels were seen in the muscle. The expression studies using polyclonal antibodies against human CARP VIII showed strong expression of CARP VIII protein in the Pur- Zebrafish CA8 is strongly expressed in the nervous system kinje cells of the zebrafish cerebellum but failed to detect any expression in the cerebrum (Fig. 3C and E). To confirm the presence of the CA8 gene in zebrafish, we first designed primers to amplify genomic DNA (Table 1) and performed PCR analysis of genomic DNA from six different AB strains of zebrafish. We analyzed the expression of the CA8 Zebrafish CA8 mRNA is expressed during gene from total mRNA of 0 day post fertilization (dpf) to early development 5 dpf larvae and confirmed the presence of CA8 gene in all To knockdown the CA8 gene using antisense morpholino developing larvae (Fig. 3A). oligonucleotides (MOs), we first checked the expression Next, we studied the expression pattern of CA8 mRNA in 12 pattern of the CA8 gene in 1–5 dpf larvae. After isolating different tissues of adult zebrafish by RT-qPCR. CA8 mRNA total mRNA, we pooled the RNA at each time point from 420 Human Molecular Genetics, 2013, Vol. 22, No. 3 Downloaded from http://hmg.oxfordjournals.org/ at Tampere University Library. Department of Health Sciences on May 19, 2014

Figure 2. Alignment of human, mouse and zebrafish CARP VIII sequences. (A) The alignment of the amino acid sequences of CARP VIII. The zebrafish sequence lacks seven glutamic acid (E) residues at N-terminal region. The region responsible for binding to IP3R1 (amino acids 45–290) is highly conserved. The amino acids substituted in homozygous recessive human patients are indicated with arrows. (B) The 3D structure of CARP VIII and coevolution analysis. The crystallographic structure of human CARP VIII (2W2J) (26) was retrieved from PDB (27) and visualized with the PyMOL Molecular Graphics System (Version 1.3.1, Schro¨dinger, LLC). Arrows indicate two human disease-causing variation sites: G162 in red, S100 in white. Residues 23–44 in gray are not included in the minimal ITPR1-binding region. Residues 1–22 are missing from the structure. The other colors identify residues that correlate strongly with at least one residue in the CARP VIII binding region of ITPR1 (1368–1649 in human ITPR1 isoform 1), with different colors for various ranges of correlation coefficient: cyan (0.92 ...0.97), yellow (0.97 ...0.980) and magenta (0.98 ...1). Human Molecular Genetics, 2013, Vol. 22, No. 3 421 ′ ′ 1–5 dpf of zebrafish larvae. Next, we performed reverse ′ ′ transcriptase-PCR (RT-PCR) to synthesize the cDNA using the primers designed based on the D. rerio ortholog found in the Ensembl database, targeting a 100 bp region of CA8 gene (Table 1). The expression of CA8 mRNA was observed in all five time points in 1–5 dpf larvae (Fig. 3A). To determine if the CA8 gene product is required for embryonic development, we first checked its mRNA expression during development. The relative expression levels of CA8 mRNA from 0 h post fertilization (hpf) to 168 hpf are shown in Supplementary Material, Figure S3A. The high level of Downloaded from CA8 mRNA at 0 hpf suggested that it is important during early development and that the CA8 transcript is of maternal AGGATCATGCTGAAATCCAAATCTG3 GCGCCATTGCTTTGCAAGAATTG 3 CGTGGAGAGAAGATTGAGTTGCCAT 3 CCTCTTACCTCAGTTACAATTTATA3 ′ ′ ′ ′ 5 5 origin. The expression of CA8 mRNA throughout the analysis suggests that the CA8 gene is required for embryonic develop-

ment in zebraﬁsh. http://hmg.oxfordjournals.org/

Developmental expression of itpr1a mRNA is similar to CA8 mRNA expression Based on the expression analysis of CA8 mRNA using RT-qPCR, CA8 mRNA was of maternal origin and was

antisense MO1antisense MO2 5 5 present in zebraﬁsh larvae at the beginning of development. It has been suggested that CARP VIII interacts with ITPR1 CA8 CA8 RC MO p53 MO sequence

and functions as a cytosolic calcium signaling modulator by at Tampere University Library. Department of Health Sciences on May 19, 2014 reducing the sensitivity of IP3 to ITPR1 (15). If this function ′ ′

′ is relevant during embryogenesis, we hypothesized that ITPR1

′ should also be expressed early in development, similar to CA8 mRNA. Out of the two ITPR1 paralogs, itpr1a and itpr1b, our coevolution analyses suggested that the itpr1a gene product is more likely to interact with CARP VIII. We, therefore, analyzed the expression of itpr1a mRNA at different time points during early embryonic development. After isolating total mRNA, we synthesized cDNA and then analyzed the expression of itpr1a mRNA using RT-qPCR with primers designed according to the zebraﬁsh sequence ENSDART00000149019 from the Ensembl database. TTAAGCCAAACTGAGACAGG3 ATGGCCTTCAGACCAAGATG 3 GGTTGGGTCGGTCTAAAGTTGT3 CGATAACCCCGGGTGAATG3 ′ ′ ′ ′

5 5 5 5 The relative expression pattern of itpr1a mRNA from 0 hpf to 168 hpf is shown in Supplementary Material, Figure S3B. A high level of itpr1a mRNA at 0 hpf suggested that itpr1a ′ mRNA is of maternal origin, similar to CA8 mRNA, and it ′ ′

′ is most likely required during embryonic development along with CA8. As shown in Supplementary Material, Figure S3A and B, both itpr1a and CA8 mRNA levels show parallel changes during the initial phase of zebraﬁsh embryogenesis, perhaps due to a functional interplay between these proteins in the developmental processes.

Knockdown of CA8 causes developmental defects in zebraﬁsh larvae

GTATTGTGATACATCAGTTT3 TTCACTGGGGCAAAGAAAAC3 CCCATGTTAAAGGAGCAGAGCTT3 CCTGGGAGCTTTCAATGTTTG3 Gene-speciﬁc antisense MOs have been widely used to sup- ′ ′ ′ ′ press the expression of genes in zebraﬁsh larvae. We designed two MOs (MO1 and MO2) (see ‘Materials and Methods’), MO1 was expected to block the translation of the CA8 gene and MO2 was designed to block the splice junction at intron

Primer sequences for PCR, RT-PCR, RT-qPCR and sequences for antisense morpholinos 3 and exon 4, as shown in Figure 4A. The CA8 antisense MO1 targets a sequence that includes the ATG start codon (from 215 to +10 relative to ATG) that inhibits the transla- Primer nameCA8 G-DNA PCR Forward 5 primer Reverse primer Morpholino sequence name MO sequence Table 1. CA8 RT-PCRCA8 RT-qPCRITPR1- RT-qPCR 5 5 5 tion of CA8 mRNA. The MO2 targets exon 4 resulting in 422 Human Molecular Genetics, 2013, Vol. 22, No. 3 Downloaded from http://hmg.oxfordjournals.org/ at Tampere University Library. Department of Health Sciences on May 19, 2014

Figure 3. Expression analysis of CA8 mRNA and the CARP VIII protein. (A) CA8 mRNA is expressed throughout the development of zebrafish larvae. RT-PCR amplification of total mRNA from 1 to 5 dpf zebrafish larvae is shown. (B) The expression of CARP VIII mRNA (NM_001017571.1) in different tissues in adult zebrafish as determined using RT-qPCR. All values are expressed relative to the expression of mRNA in the brain, which was assigned a relative value of 1, and the values were normalized by inclusion of an internal control gene beta actin according to the pfuffl (24). (C and D) CARP VIII is expressed in the Purkinje cells of cerebellum. Immunohistochemical analysis was carried out using polyclonal antibodies raised against human CARP VIII. (E) CARP VIII is not detected in the cerebrum by immunostaining. (F) A negative staining control using rabbit non-specific IgG antibody instead of the CARP VIII antibody (C, E and F, magni- fication 20× and D 40×). expression of shorter length CA8 gene. These antisense MOs polyclonal antibodies raised against 100 amino acids of the N- were used to analyze the role of CA8 in zebrafish embryonic terminal domain of human CARP VIII detected a 32 kDa development. Uninjected larvae and those injected with band. In contrast, lysates from the larvae injected with random control (RC) morpholinos were used as controls for 125 mM CA8-MO1 did not show any band after 3 dpf the phenotypic analysis of 1–5 dpf larvae. (Fig. 4B). After 5 dpf, the lysates from larvae injected with We used western blot analysis to determine the expression 125 mM CA8-MO1 showed a lower intensity 32 kDa band, of CARP VIII and RT-PCR analysis of CA8 mRNA to when compared with the wild-type embryo lysates (Fig. 4C). ensure the effectiveness of the CA8 antisense MO1 and This indicates that the CA8 antisense MO1 suppresses MO2. As shown in the Figure 4B and C, western blotting of CARP VIII expression in 3 dpf zebrafish larvae. Similarly, the 3 dpf uninjected (wild type) zebrafish lysates using analysis of mRNA from 5 dpf morphant embryos injected Human Molecular Genetics, 2013, Vol. 22, No. 3 423 Downloaded from http://hmg.oxfordjournals.org/

Figure 4. Western blotting and mRNA analysis of wild-type and CA8 morphant larvae. (A) Schematic presentation of CA8 mRNA showing the sites of translational blocking at the beginning of exon one (arrow) and splice site blocking at the junction of intron 3 and exon 4 as indicated by an arrow. (B) Western blotting analysis of lysates from 3 dpf zebrafish larvae microinjected with 125 mM CA8-MO1 indicates efficient suppression of CARP VIII protein expression. (C) The western blot of samples extracted from 5 dpf morphant larvae microinjected with 125 mM CA8-MO1 morpholinos shows the reappearance of the CARP VIII protein at the end of 5 dpf. (D) RT-PCR analysis of mRNA from morphant and control larvae analyzing the changes in the mRNA length of CA8 gene caused by MO2. at Tampere University Library. Department of Health Sciences on May 19, 2014 with 250 mM CA8-MO2 showed a shorter length mRNA as a 75 mM or higher CA8-MO1 (Fig. 5C, Table 2). There was no lower intensity band in addition to normal length mRNA, sug- difference between the phenotype defects or mortality rates gesting a partial knockdown of CA8 with MO2, as shown in of larvae injected with CA8-MO1 alone or together with Figure 4D. p53-MOs (Fig. 5C and Table 2). The western blot results Larvae injected with 125 mM CA8-MO1 showed abnormal (Fig. 4B and C) suggested that the concentration required changes in the head as early as 9 hpf (data not shown). The for the complete knockdown of CA8 gene was 125 mM,at 1 dpf zebrafish larvae injected with 125 mM CA8-MO1 which level the morphant larvae showed severe phenotypic showed defects in the head with a fragile and more easily defects. Therefore, 125 mM MO1 was the optimal effective breakable body, curved tail, small eye size and pericardial concentration used for the most of the experiments in this edema (Fig. 5A). As development progressed, the abnormal- study. However, the phenotype of 5 dpf larvae injected with ities became more prominent; shortened tail, curved body 125 mM concentration of MO2 was similar to the phenotype axis, absence of swim bladder and otolith vesicles were of 6 mM MO1-injected larvae and showed an abnormal swim observed, when compared with the control larvae (Fig. 5A). pattern (Supplementary Material, Movie S5). Interestingly, embryos injected with MO2 showed a less severe phenotype, when compared to the phenotype produced by MO1 (Fig. 4B). In dose–response analysis with the CA8 morphant zebrafish have increased neuronal CA8-MO1, larvae showed no apparent phenotypic changes at cell death in the cerebellum low concentrations of CA8-MO1, whereas the increased concen- Expression analyses of the CA8 gene in zebrafish tissues trations of CA8-MO1 led to severe phenotypic defects with showed a widespread distribution of CA8 mRNA (Fig. 3B). increased mortality (Supplementary Material, Figure S2A–G, The highest expression was observed in the brain, with and Table 2). The injection of 2 mM CA8-MO1 per embryo lower levels of expression in other organs, suggesting an im- did not produce any visible defects or changes in the mortality portant function in several tissues. We studied morphologic rate, when compared with the uninjected controls (Supplemen- changes in larvae injected with 125 mM CA8-MO1 and com- tary Material, Figure S4B, Table 2); however, the swimming pared the findings with control larvae injected with RC MOs pattern was abnormal, suggesting that 2 mM CA8-MO1 is the and p53 MOs. We made semithin (10 mm) sections of 5 dpf minimum dose required to produce the knockdown effect of control larvae and larvae injected with 125 mM CA8-MO1 CA8 (Supplementary Material, Movie S2). The next higher and stained them with hematoxylin and eosin. The 125 mM concentration examined was 6 mM CA8-MO1, which showed CA8-MO1-injected larvae showed gross morphologic a slightly visible functional defect during swimming (Supple- changes in the cerebellar region with a reduction in the size mentary Material, Figure S4C and S5B) and a mortality rate of the cerebellum (Fig. 6H). Similarly, there was a clear differ- of 10.6% (Fig. 5C and Table 2). The larvae injected with con- ence between the somites of control larvae and those of the centrations of 10 mM,75mM, 125 mM, 250 mM and 500 mM CA8-MO-injected morphant larvae. The CA8-MO1-injected CA8-MO1 showed moderate to severe phenotypic defects, zebrafish showed abnormal muscle development, when com- and the mortality rate increased in larvae injected with pared with the control zebrafish larvae (data not shown). 424 Human Molecular Genetics, 2013, Vol. 22, No. 3 Downloaded from http://hmg.oxfordjournals.org/ at Tampere University Library. Department of Health Sciences on May 19, 2014

Figure 5. The CA8 gene product is essential for the normal embryogenesis of zebrafish. Developmental images are shown over the period of 0–5 dpf of zebrafish larvae injected with 125 mM translational blocking antisense morpholinos (MO1) for the CA8 gene when compared with normal (uninjected) larvae. (A) The top row of images show lateral views of 0–5 dpf uninjected zebrafish larvae with the gradual, normal development of organs, the disappearance of yolk sac and the appearance of swim bladder at the end of 5 dpf. The bottom row of images show lateral views of CA8 morphants embryo from 0 to 5 dpf showing morphologic changes at 24 hpf with abnormal shaped head, tail and yolk sac and the beginning of dilation of the pericardium. (B) The 5 dpf larvae injected with 250 mM of CA8-MO2 (splice site blocking) showing a defective phenotype. (C) The percent mortality in CA8 morphant larvae at 24 hpf. The histogram shows the percentage of dead larvae in each group injected with different concentrations of CA8-MO1 when compared with the RC-injected and uninjected zebrafish larvae. The mortality rate was significantly higher (P , 0.001) in the larvae injected with greater than 10 mM CA8-MO1. There was no difference in mortality rates of the larvae injected with p53-MOs along with CA8-MOs and larvae injected with CA8-MO1 alone.

The TdT-UTP nick end labeling (TUNEL) assay showed cells region of the zebrafish injected with 125 mM CA8-MO1 using undergoing apoptosis in the head region and periphery of tail transmission electron microscopy to detect any ultrastructural region in the larvae injected with 125 mM CA8-MO1, whereas changes occurring as a consequence of knockdown of the CA8 there was no detectable signal for apoptosis in the wild-type gene. The only significant change at the ultrastructural level larvae (Fig. 6A–D). was the increased neuronal cell death (the apoptotic changes After finding the morphologic changes in the cerebellum by included a condensed nucleus, fragmented mitochondrial pro- histologic staining and light microscopy and cell death in the files and debris of dead cells) that was observed in samples head region by the TUNEL assay, we analyzed the cerebellar from the MO1-injected zebrafish, when compared with the Human Molecular Genetics, 2013, Vol. 22, No. 3 425

Table 2. Phenotypes of 24 hpf zebraﬁsh larvae knocked down with different concentrations of CA8 antisense morpholinos

Morpholino (mM) Normal Mild Moderate Severe Dead Total

CA8 MO 250a 0 (0) 0 (0) 15 (10) 69 (46) 66 (44) 150 CA8 MO 250 + p53 375a 0 (0) 0 (0) 14 (9.3) 71 (47.3) 68 (45.3) 150 CA8 MO 125b 11 (2.2) 0 (0) 8 (1.6) 311 (62.2) 162 (32.4) 500 CA8 MO 125 + p53187.5b 13 (2.6) 7 (1.4) 9 (1.8) 320 (64) 151 (30.2) 500 CA8 MO 10c 8 (5.3) 18 (12) 93 (62) 7 (4.6) 24 (16) 150 CA8 MO 6c 20 (13.3) 104 (69.3) 11 (7.3) 23 (15.3) 16 (10.6) 150 CA8 MO 2c 132 (88) 12 (8) 0 (3.3) 0 (15.3) 6 (4.0) 150 RC MOb468 (93.6) 0 (0) 0 (3.3) 0 (15.3) 32 (6.4) 500 Uninjectedb 472 (94.4) 0 (0) 5 (1.0) 23 (4.6) 28 (5.6) 500 Downloaded from

The phenotypic data were obtained from total of 3a,10b and 2c independent sets of morpholino injections with a minimum of 50 zebrafish larvae per group. The total number of larvae examined for each phenotypic class is shown as percentage of total number of larvae studied for each concentration of morpholinos. RC morpholino injections were made with either RC-MO alone or RC-MOs together with p53 MOs (one-and-a-half times the RC-MO concentration). Larvae injected with 2 mM concentrations of MOs had a phenotype similar to uninjected larvae but showed abnormal swim patterns (Supplementary Material, Figure S2, and Movie S2). http://hmg.oxfordjournals.org/ wild-type larvae (Fig. 6E and F). There was no difference compared with the uninjected larvae, larvae injected with between the morphant larvae injected with CA8-MO1s alone 6 mM concentrations of CA8-MO1s moved considerably or in combination with p53-MOs. slower, showed an increased turning angle and mostly swam along the periphery of the Petri dish. Additionally, the larvae injected with 6 mM CA8-MO1s showed a pronounced CA8 morphant zebrafish larvae have altered difficulty in balancing the body while swimming, when com- movement patterns pared with the wild-type larvae (Supplementary Material, at Tampere University Library. Department of Health Sciences on May 19, 2014 Because of the ataxia and lifelong gait disorder in wdl mice and Figure S5A and B and Movies S1 and S2). Moreover, the ataxia and quadrupedal gait in human patients (1–3)dueto larvae injected with 10 mM CA8-MO1s had severely restricted spontaneously occurring mutations in the CA8 gene, we ana- movement and showed occasional displacement at the corners lyzed whether the CA8 knockdown changed the swimming be- of the Petri dish with great difficulty in balancing themselves havior of 5 dpf zebrafish larvae. We compared spontaneous (Supplementary Material, Figure S4 right and Movie S4). The swimming behavior of 5 dpf control larvae (uninjected), larvae injected with 125 mM CA8-MO1s, which had severe larvae injected with RC morpholinos and larvae injected with phenotypic defects, exhibited occasional sideways turning different concentrations of antisense CA8-MO1. We expected and did not show any displacement. The larvae injected with that the larvae with body and tail abnormalities, induced by 125 mM CA8-MO2, which had phenotypically normal appear- CA8 antisense morpholinos with concentrations above 6 mM, ance of the body, showed an abnormal swim pattern similar to would show abnormal swimming behavior. To test this hypoth- the 6 mM CA8-MO1-injected larvae (Supplementary Material, esis, we injected the larvae with minimum concentrations of Movie S5). 2and6mM CA8-MO1. With these concentrations, the 5 dpf larvae showed normal body and tail development, similar to RC-injected and uninjected larvae (Supplementary Material, DISCUSSION Figure S4A, B, and C). For each experiment, five different groups (uninjected and injected with CA8-MO1 concentrations Here, we show that the knockdown of the CA8 gene, which is of 2 mM,6mM, 10 and 100 mM) were analyzed for movement responsible for ataxia, gait disorder and mental retardation in patterns. human patients and mice, results in an abnormal movement We tracked the movement patterns of the 5 dpf larvae under pattern in zebrafish, mimicking the movement disorder in the microscope in a 35 mm × 15 mm transparent polypropyl- humans and mice. Our observations suggest that the suppres- ene Petri dish filled with embryo medium containing a sion of CA8 expression is responsible for the abnormal move- minimum of five zebrafish larvae. After placing the Petri ment pattern in zebrafish larvae: (i) injection of CA8 antisense dish under the microscope (Carl Zeiss Lumar V.12MicroIma- MO1s at 2 mM concentration shows abnormal movement ging GmbH and software AxioVision versions 4.7 and 4.8.), without any morphologic phenotypic defects, (ii) RC MOs the larvae were allowed to settle for at least 2 min, and the do not show any change in the behavior or morphology even movement of larvae was observed for 10 min to trace the at very high (125 mM) concentrations and (iii) the abnormal pattern of movement (shown schematically in Fig. 7A). The movement and structural changes in the larvae were dependent percentage of movement in CA8-MO1-injected larvae when on the concentration of the CA8-antisense morpholinos compared with the control larvae was analyzed for each injected. group (Fig. 7B). A 30 s movie of the movement patterns Evolutionarily conserved genes generally have important was also recorded and can be seen in Supplementary data biologic functions, manifested through high selection pressure. (Supplementary Material, Movies S1–S4). The larvae injected The high conservation of CARP VIII across the species, with 2 mM CA8-MO1 had a slightly abnormal pattern of swim- exceeding even the conservation of enzymatically active ming, when compared with the wild-type larvae. When CAs, and the very specific expression pattern in the brain 426 Human Molecular Genetics, 2013, Vol. 22, No. 3 Downloaded from http://hmg.oxfordjournals.org/ at Tampere University Library. Department of Health Sciences on May 19, 2014

Figure 6. TUNEL assay, electron microscopy and histochemical staining. (A) At 5 dpf, prominent apoptosis was observed in head region of zebrafish larvae with the CA8 gene knocked down. (B) Apoptosis was also observed at the periphery of the tail region in larvae knocked down for CA8 gene. (C and D) There was no apoptosis in control larvae in either the head region C or tail region D. (E) A micrograph of control (uninjected) 5 dpf zebrafish larvae inside a normal neuronal cell in the cerebellum. (F) A micrograph of a 5 dpf zebrafish embryo injected with 125 mM CA8-MO1 showing inside of a neuronal cell undergoing apoptosis in the cerebellum. Abbreviations: M, mitochondria; ER, endoplasmic reticulum and Nuc, nucleus. (G) A sagittal section of the head region showing normal morphology of the cerebellar region, 20×.(H) A sagittal section of the head region showing abnormal morphology of the cerebellar region, 20×. Human Molecular Genetics, 2013, Vol. 22, No. 3 427 Downloaded from http://hmg.oxfordjournals.org/ at Tampere University Library. Department of Health Sciences on May 19, 2014

Figure 7. Swim patterns of control and CA8 morphant larvae. The trajectories of the 5 dpf larvae injected with different concentrations of CA8-MO1 when compared with uninjected 5 dpf larvae. Zebrafish larvae at 5 dpf show a concentration-dependent increase in abnormal swimming patterns when compared with the uninjected 5 dpf larvae. The swimming trajectories of a minimum of five larvae were observed for 10 min in a 15 × 30 mm sterile petri dish in embryo medium under a Zeiss microscope. The larvae were initially allowed to rest for 2 min after the transfer of larvae to the petri dish and the swim pattern was studied for 10 min. The comparison of swimming trajectories of uninjected larvae with larvae injected with different concentration of CA8-MO1 (A). The displacement of the larvae was traced for 10 min using Zeiss microscope. The percent displacement of the morphant larvae injected with different concentrations of CA8-MOs compared with uninjected larvae (B). Half-minute sample movies of swim patterns of uninjected larvae and those injected with three different concentrations of CA8 antisense MOs (2, 6 and 10 mM) can be seen in additional Supplementary movie files. indicate that CARP VIII has a very important biologic development. We focused on the developmental and behavior- function (9). al consequences of the knockdown of the CA8 gene and on CARP VIII is an enzymatically inactive member of a-CA brain ultrastructure in the embryonic development of zebra- gene family, similar to CARP X and CARP XI, and its fish. exact physiologic role is only starting to emerge. Previous The RT-qPCR analysis of the relative expression of CA8 reports suggest that CARP VIII is abundantly expressed in mRNA in 0 hpf–168 hpf showed the expression pattern human and mouse brain, especially in cerebellar Purkinje throughout the developmental period, with peaks at 0, 24 cells, and it controls intracellular calcium signaling by and 120 hpf. The presence of a high level of maternal CA8 binding to ITPR1 (14). A report on wdl mice, which have a mRNA at 0 hpf shows that it is required early in embryonic 19 bp deletion in the Car8 gene, demonstrated clear ataxia development. and dystonia with a lifelong gait disorder (3). Recent studies In a previous mouse study (16), the expression of Car8 have also shown that spontaneously occurring mutations in mRNA was determined by in situ hybridization during embry- the CA8 gene lead to similar defects in coordination and onic development. At 9.5 days of gestation, Car8 mRNA is mental retardation in human patients (1,2). Apart from these expressed in a variety of organs, such as liver, bronchial reports, there are neither studies on the role of CARP VIII, arches, neuroepithelium and developing myocardium. As de- nor do any knockout or knockdown vertebrate models exist velopment progressed, the expression became restricted to for studying its physiologic role in the embryonic development brain, liver, lung, heart, gut, thymus and the epithelium cover- of vertebrates. ing the head and the oronasal cavity. Unfortunately, there were The objective of this study was to determine in a zebrafish no results prior to 9.5 days of gestation. In another mouse knockdown model whether the CA8 gene product is required study (15), the expression was analyzed in different tissues for normal embryonic development, especially in brain at four different gestational days by RT-PCR and northern 428 Human Molecular Genetics, 2013, Vol. 22, No. 3 blot analysis (7–17 days after fertilization). Expression of CA8-MOs was further confirmed by western blotting with Car8 mRNA was found at 11 days of gestation by RT-PCR polyclonal antibodies against the CARP VIII protein and ana- but only at 15 days of gestation by northern blot analysis. lysis of mRNA length from CA8-MO2 morphant embryos. The developmental expression analysis in human brain sug- The possible off-target effects due to antisense MOs were gested that the CA8 gene and CARP VIII protein are expressed excluded by injecting RC morpholinos that produced normal at 84 days of gestation in the brain with discrepant findings larvae, similar to uninjected ones, which again supported the between the results of northern blot and immunohistochemical specificity of CA8-MOs. The off-target effects of antisense analysis (8). CA8/Car8 expression during development would morpholinos occasionally lead to apoptosis. To suppress the need to be reinvestigated in a mammalian model by a more possible off-target effects of CA8-MOs, if any, we also sensitive method, such as RT-qPCR. injected p53-MOs, which excluded the disruption of myo- Similar to our previous study on the expression of Car8 tomes and pericardial edema as non-specific effects of mor- Downloaded from mRNA in a panel of mouse tissues, we analyzed the expres- pholino studies (17). sion pattern of CA8 mRNA by RT-qPCR in 12 different zebra- The specificity of the morpholinos was confirmed by fish tissues. The expression analysis showed predominant western blotting of lysates from 3 dpf larvae injected with expression of CA8 mRNA in the brain (Fig. 3B) similar to 125 mM CA8-MO1s and the analysis of mRNA length from

the mouse Car8 and human CA8 expression patterns. This ex- MO2 morphant larvae. Although these samples showed no http://hmg.oxfordjournals.org/ pression pattern suggests that it is required in the early devel- 32 kDa polypeptide band corresponding to CARP VIII, the opment and function in the zebraﬁsh brain. High expression analysis of lysates after 5 dpf (injected with 125 mM MOs) levels were also found in zebraﬁsh kidneys, heart, eyes, tail showed a faint band at 32 kDa, suggesting that the effect of and skin. Low expression levels were observed in male and CA8-MO1 was diminishing. However, the presence of female gonads, liver, gills and bowel, and a weak signal was CARP VIII protein after 5 dpf did not rescue the phenotypic found in the muscle in this study. The expression patterns of defects that occurred early in the development. In a previous CA8 in these other tissues were different from mouse, with a study, knockdown of the huntingtin protein produced defective high level of expression in kidneys, eyes and heart and male phenotypes in 3 dpf larvae, but contrary to our results with

and female gonads in the zebrafish; we have currently no ex- CARP VIII, the huntingtin phenotypes were reversed after at Tampere University Library. Department of Health Sciences on May 19, 2014 planation for this unique pattern of expression in zebrafish. 5 dpf when the MOs could no longer completely suppress Similar to the predominant expression of the CARP VIII the translation (18). protein in cerebellar Purkinje cells in human and mice by Our study suggests that CARP VIII protein is required for immunohistochemistry (8,9), our immunohistochemistry normal development during early embryogenesis and that results showed an intense signal for the CARP VIII protein changes occurring early in development resulting from insuf- in Purkinje cells of cerebellum. This localization agrees with ficient CARP VIII protein are not reversible, even if the the motor coordination defects observed in the phenotypes, protein reappears later in the development. both in our study and in previous examples from mice and CARP VIII is known to interact with ITPR1 and to modu- men (1–3). late the calcium ion release from intracellular reserves. Both The zebrafish CARP VIII protein has an unusually high CARP VIII and ITPR1 are expressed abundantly in the Pur- level of sequence identity with human CARP VIII (10), yet kinje cells of mice cerebellum (14). Because the ITPR1 gene the functional significance of CARP VIII in vertebrate devel- has been duplicated during the evolution of fish, we performed opment is not known. We hypothesized that zebrafish CARP a coevolution test (analysis of correlated mutations in two VIII might play a critical role during embryogenesis because genes) to evaluate which of the fish orthologs would more (i) CARP VIII is highly conserved, (ii) it is expressed within likely have retained the interaction with CARP VIII. We ana- the Purkinje cells of cerebellum similar to humans and mice lyzed the correlations in sequence variation between the CA8 and (iii) a high amount of its mRNA of maternal origin is and ITPR1 gene products in 31 vertebrate species, using either present during the earliest stages of embryogenesis. Therefore, itpr1a or itpr1b sequences from six fish species in two parallel we further hypothesized that knockdown of the CA8 gene analyses. The correlations were stronger for itpr1a, which using antisense morpholinos might result in an abnormal indicates that itpr1a is more likely to have coevolved with phenotype in zebrafish larvae. CA8 in fishes. Coevolution means that changes in one gene We knocked the CA8 gene down by injection of translation- product are reflected as changes in the other to retain a func- blocking and splice site-blocking antisense MOs at the 1–2 tional interaction; therefore, we suggest that itpr1a is the main cell stage in zebrafish larvae. The larvae revealed phenotypes binding partner of CARP VIII in fishes, whereas the itpr1b from very mild (without apparent morphologic defects with gene product diverges to perform other functions. 2 mM CA8-MO1) to a severe phenotype (500 mM CA8-MO1) We studied the coevolution of residues in CARP VIII in the with morphologic defects such as a short curved tail, abnormal 3D structure and noticed a cluster of residues (150–157) in the head shape, small eyes, pericardial edema, abnormal otolith vicinity of the known human disease-causing variation sites. sacs, absence of swim bladder, fragile body and defects in We suggest that the binding site to ITPR1 would be at or somites. The abnormal head shape was observed as early as near this region. The coevolution analysis also narrows 9 hpf during development (data not shown). The specificity down our estimate of the CARP VIII binding region in of the CA8-MOs was indirectly confirmed by repeatedly repro- ITPR1 to amino acids 1437 to 1630 in human isoform 1, cor- ducible phenotypes obtained by injection of translation- responding to 1451 to 1644 in mouse isoform 1. blocking and splice site-blocking morpholinos specific for We measured itpr1a mRNA levels during zebrafish embry- CA8 gene. The specificity of the knockdown effect by onic development from 0 hpf to 168 hpf. Our results show that Human Molecular Genetics, 2013, Vol. 22, No. 3 429 the temporal expression pattern of itpr1a is very similar to that reports of human disease-causing CA8 mutations (1,2)It of CA8, which further supports their functional association. remains to be seen if more severe mutations in CA8 in The phenotype in the morphant larvae is most likely due to humans lead to viable individuals. By allowing partial knock- the improper modulation of ITPR1 function for calcium sig- down with lower concentrations of MOs, the model presented naling in the brain in the absence of CARP VIII. It has been in this study helps us to understand the phenotypic spectrum hypothesized that the binding of CARP VIII to ITPR1 in the we may see in human patients with defects in the CA8 gene. regulatory region (Supplementary Material, Figure S2) In summary, we describe a zebrafish model with an abnor- reduces the sensitivity of IP3 to its receptor ITPR1, and mal movement pattern resulting from the knockdown of the hence, the controlled calcium release from intracellular CA8 gene. This is similar to defects in CARP VIII, which reserves. In the absence of CARP VIII, the sensitivity of lead to ataxia, mental retardation and quadrupedal gait in ITPR1 to IP3 increases and excessive calcium is released human patients and ataxia and lifelong gait disorder in wdl Downloaded from from intracellular reserves (15,19). mice. In addition, we provide evidence that the CA8 gene The ataxia and mental retardation observed in human product is indispensable in early embryonic development in patients with CA8 variations are also likely due to disruption zebrafish, particularly in the brain and eye. Our study also sug- of the ITPR1 interaction. Interestingly, mutations in ITPR1 gests that this phenomenon is conserved across vertebrate

lead to ataxia in mice, and mutations in ITPR1 have been iden- species. This novel zebraﬁsh model is available for further http://hmg.oxfordjournals.org/ tiﬁed in spinocerebellar ataxia 15 in humans (20,21). Thus, the investigations of the mechanisms of CARP VIII-related involvement of CA8 should be considered in the diagnosis of ataxia and mental retardation in humans. unknown forms of ataxia and mental retardation in humans. Because many CAs facilitate ion transport and bind to ion transporters (22) and CARP VIII is involved in ion channel MATERIALS AND METHODS regulation, we have arrived at a hypothesis for the unknown Bioinformatic analysis of the CA8 gene and CARP VIII functions of other highly conserved CARPs. We propose protein that CARP X and CARP XI would be modulators of ion trans-

port similar to CARP VIII. Our search for CARP X targets is The zebrafish (ID: ENSDARP00000057097), mouse (ID: at Tampere University Library. Department of Health Sciences on May 19, 2014 underway. ENSMUSP00000095891) and human (ENSP00000314407) Studies on the cerebellum in wdl mice, with a 19 bp deletion CARP VIII protein sequences were obtained from the in Car8 gene, suggest a critical role for CARP VIII in synap- Ensembl database. Multiple sequence alignments and the count- togenesis and/or maintenance of proper synaptic morphology ing of percent identity of amino acids were conducted for all the and function in the cerebellum (20). Previous reports on a three CARP VIII sequences using the ClustalW program CA8 CA8 mutation in members of an Iraqi family did not provide transcripts for zebrafish (ID: ENSDART00000057098), mouse any data on possible morphologic changes of the cerebellum (ID: ENSMUST00000098290) and human (transcript ID: (2). However, another report on a human CA8 mutation ENST00000317995) were from Ensembl, and the organization revealed a significant loss in the volume of cerebellum propor- of the exon structure and comparative analysis of all the three tional to the age of the patients with ataxia and mental retard- transcripts was performed. ation (1). In this zebrafish study, we noted obvious external malfor- mations and a lack of proper head development at the time Coevolution analysis point of neuronal differentiation (1 dpf). In addition, we The vertebrate ortholog protein sequences of human CARP detected increased neuronal apoptosis in the cerebellum of VIII and ITPR1 proteins were obtained from Ensembl. The the 5 dpf CA8 knockdown larvae. Histologic examination of ITPR1 sequences were edited to contain only the region semithin sections of the 5 dpf larvae revealed gross morpho- mapped for CARP VIII binding (12), plus four residues on logic change in the cerebellar region with a reduction in the the N-terminal and seven residues on the C-terminal side (cor- size of cerebellum in the brain and alterations in somite struc- responding to 1368–1649 in human ITPR1 isoform 1). Like- tures and muscle fibers. Moreover, electron microscopy wise, the CARP VIII sequences were shortened to the studies showed increased neuronal cell death in the cerebellum minimal ITPR1-binding fragment 45–291 (human number- in 5 dpf zebrafish larvae, mimicking the phenotype found in ing). Only complete sequences were retained, and the se- human patients. quence sets were reduced so that we had an identical list of We showed that the phenotypic changes became more species for both proteins. The CARP VIII alignment consisted prominent as the concentration of CA8 antisense MOs was of 31 sequences, whereas the ITPR1 alignment included 37 increased. Our data also indicate that the loss of the CA8 sequences, due to an early duplication in the fish lineages, gene leads to a swim pattern abnormality similar to the which has led to two paralogs for ITPR1 genes in fish ataxia and gait disorder in wdl mice and ataxia in human genomes. A phylogenetic tree was reconstructed using the patients with CA8 mutations (1–3). The severe phenotype CARP VIII alignment with the neighbor-joining method, observed with the 125 mM concentration of CA8-MO1 is rooting by using the fish sequences as the outgroup. because this concentration of MOs targets both zygotic and Two alignments were built from the ITPR1 sequences so maternal CA8 mRNA and completely suppresses CA8 that the 25 tetrapod species were present in both, whereas mRNA in zebrafish larvae. The zebrafish knockdown model the 12 sequences from 6 fish species were divided. The ortho- with a high concentration of MOs resembles a null mutant logs of D. rerio itpr1a were included in one alignment phenotype, in contrast to the mutations found in the two (‘clade1’) and the orthologs of itpr1b in another alignment 430 Human Molecular Genetics, 2013, Vol. 22, No. 3

(‘clade2’). The two alignments of ITPR1 from the 31 species from Ensembl (ENSDART00000057097) database (Supple- were analyzed separately for coevolution with the CARP VIII mentary Material, Figure S1). sequences. The CAPS program (21) (PMID: 17 005 535) was used with the sequence alignments and the phylogenetic tree to identify Real-time quantitative PCR the likelihood of coevolution between sites of CARP VIII and RT-PCR was performed using 0.1–5 mg of total RNA to syn- ITPR1 proteins. thesize the first strand cDNA using a First Strand cDNA Syn- thesis kit (High-Capacity cDNA Reverse Transcription Kits, Applied Biosystems, Foster City, CA, USA) with random Zebrafish husbandry for experiments primers and M-MuLV Reverse transcriptase according to the Downloaded from Wild-type zebrafish of the AB strain were maintained at protocol recommended by the manufacturer. Real-time 28.58C under standard conditions (23). Larvae were collected qPCR primers were designed based on the complete cDNA se- from the breeder tanks with a sieve, rinsed with embryonic quence taken from Ensembl (ENSDART00000057097), using medium (Sarsted, Nu¨mbrecht, Germany) and placed into the program Primer Expressw Software v2.0 (Applied Biosys- Petri dishes (24). The larvae were kept in Petri dishes in em- tems) (Table 1).

Real-time qPCR was performed using a SYBR Green PCR http://hmg.oxfordjournals.org/ bryonic medium supplemented with 1-phenyl 2-thiourea TM (Sigma-Aldrich) at 28.58C until they were used in experi- Master Mix Kit in an ABI PRISM 7000 Detection System , ments. The maximum number of larvae on a 9 cm diameter according to the manufacturer’s instructions (Applied Biosys- Petri dish was 50. The embryonic medium contained 5 mM tems). The PCR conditions consisted of an initial denaturation NaCl, 0.17 mM KCl, 0.33 mM CaCl , 0.33 mM MgSO and step at 958C for 10 min followed by 40 cycles at 958C for 15 s 2 4 (denaturation) and 608C for 1 min (elongation). The data were 10–15% Methylene Blue (Sigma-Aldrich). The embryonic TM medium in Petri dishes was replaced every day with Pasteur analyzed using the ABI PRISM 7000 SDS software pipette and larvae were kept at 28.58C. (Applied Biosystems). Each PCR reaction was performed in a total reaction volume of 15 ml containing 2 ml of ﬁrst

strand cDNA (20 ng cDNA), 1 × Power SYBR green PCR at Tampere University Library. Department of Health Sciences on May 19, 2014 TM RNA extraction Master Mix (Applied Biosystems, Foster City, CA, USA) and 0.5 mM of each primer. The final results, expressed as Total RNA was isolated from 0 to 168 hpf whole larvae at dif- the N-fold relative difference (ratio) in gene expression ferent stages of development and from different organs of the between the studied samples, internal control gene b-actin adult zebrafish. The samples used for mRNA isolation were and the relative expressions were calculated according to stabilized in RNA later (Ambion, Austin, TX, USA) immedi- Pfaffl’s equation with appropriate modifications (25). ately following collection, and the total RNA was isolated from 30 mg sample using an RNAeasyw Mini kit (Qiagen, Hilden, Germany) by following the manufacturer’s instruc- Immunolocalization of the CARP VIII protein in the adult tions. The concentration and purity of total RNA were deter- zebrafish brain mined using a Nanodrop Spectrophotometer at 260 and Wild-type adult zebrafish heads were fixed in 4% paraformal- 280 nm. dehyde (PFA) in phosphate-buffered saline (PBS) and embedded in paraffin. Fluorescent immunohistochemistry was carried out on rehydrated sections (5 mm) of normal adult zeb- Synthesis of cDNA, PCR amplification, cloning and rafish heads. The specimens were rinsed with PBS and sub- sequencing jected to immunofluorescence staining that consisted of the Total RNA was isolated from brain tissue using an RNAeasyw following steps: (i) pretreatment with 0.1% BSA in PBS Lipid Tissue extraction kit from Qiagen according to the (BSA-PBS) for 30 min, (ii) incubation for 1 h with rabbit anti- instructions. RT-PCR was performed using 0.1–5 mgof human CARP VIII antibody (Santa Cruz Biotechnology, Inc., total RNA to synthesize the first strand of cDNA using Bergheimer Heidelberg, Germany) (diluted 1:20) or normal a First Strand cDNA Synthesis kit (High-Capacity cDNA rabbit serum (diluted 1:20) in 0.1% BSA-PBS, (iii) rinsing Reverse Transcription Kits, Applied Biosystems, Foster City, three times for 5 min with BSA-PBS, (iv) incubation for 1 h CA, USA) with random primers and M-MuLV reverse tran- with 1:100 diluted Alexa Fluor 488 goat anti-rabbit IgG anti- scriptase according to the protocol recommended by the bodies (Molecular Probes, Eugene, Oregon, USA) in 0.1% manufacturer. BSA-PBS and (v) rinsing twice for 5 min with BSA-PBS PCR amplification was carried out using the forward and and once with PBS. Immunostained sections were then ana- reverse primers designed for CA8 gene in Ensembl lyzed and photographed using a Zeiss LSM 700 Confocal (Table 1). The resulting PCR product was ligated into a laser scanning microscope. plasmid vector (pcDNA3.1). The plasmid/CA8 construct was then transformed into One Shotw TOP10 competent cells (Invitrogen, Espoo, Finland), and the cells were plated on Analysis of genomic DNA from adult zebrafish and LB/ampicillin plates. The colonies were screened by colony expression of CA8 in 1–5 dpf larvae PCR for the presence of the correct insert. DNA sequencing Before designing antisense MOs for translation and splice site of four different clones was carried out. The sequences were blocking of CA8 mRNA, we checked the expression pattern aligned with ClustalW and compared with cDNA obtained during developmental stages 1–5 dpf in the larvae for the Human Molecular Genetics, 2013, Vol. 22, No. 3 431 knockdown study. Total RNA from 1 to 5 dpf larvae was used in embryo medium and embedded in 17% high molecular to synthesize the first-strand cDNA using reverse transcriptase weight methyl cellulose in 15 × 30 mm transparent polypro- (Superscript II kit; Invitrogen). The resulting cDNA was then pylene Petri dish [for taking images of the developing larvae amplified using primers for a 100 bp region of CA8 cDNA starting from Day 1 to Day 5 using a Lumar V1.12 fluores- (Table 1). To check for possible polymorphisms at the mor- cence stereomicroscope attached with a camera and with pholino target sequence in AB strain of zebrafish in our labora- 1.5× lens (Carl Zeiss MicroImaging GmbH, Go¨ttingen, tory (Table 1), we amplified PCR products for the target Germany)]. The images were analyzed with AxioVision soft- region and sequenced the PCR product from the six different ware versions 4.7 and 4.8. zebrafish individuals. Downloaded from Morpholino injections Behavioral analysis of 5 dpf CA8 knockdown zebrafish larvae Two independent (one translation blocking and second splice junction blocking) antisense MOs (GeneTools LLC, Philo- Larvae were tested for behavioral consequences due to the math, OR, USA) were designed by GeneTools to target the CA8 knockdown by measuring the swimming pattern at 5 dpf. Control (uninjected) and CA8 knockdown larvae were

CA8 translation initiation site. The random MOs that were http://hmg.oxfordjournals.org/ used as a control and p53-MOs used to suppress the expression raised in embryo medium and, after 5 dpf, screened for swim- of p53 mRNA were also obtained from Gene Tools (Table 1). ming patterns. Fully developed larvae were arrayed (5 per × The supplied MOs were resuspended in sterile water at a 1 mM well) in a 15 30 mm one-well Petri dish containing stock concentration. Immediately prior to injection, the embryo medium and placed on a light stage with a transmitted CA8-MOs were diluted to the intended concentration (500, light source. The larvae were allowed to acclimate to the dish 250, 125, 75, 10, 6 and 2 mM), and 1% phenol red (Sigma, for 2 min, and the movement patterns were observed for Poole, UK) was added to the solution to monitor injection ef- 10 min, while simultaneously tracing the movements of the ficiency. We injected 1 nl of antisense MOs into the yolk of fish. Similarly a 30 s movie was recorded for each group using a Lumar V1.12 fluorescence stereomicroscope (Carl one- to two-cell stage larvae. When p53-MOs were included at Tampere University Library. Department of Health Sciences on May 19, 2014 for injections, the concentration of p53-MOs was Zeiss MicroImaging GmbH) and AxioVision software ver- one-and-a-half times the concentration of antisense CA8-MOs. sions 4.7 and 4.8. Each experiment was repeated a minimum of three times.

Western blotting and mRNA analysis of CA8-MO-injected zebrafish larvae Histochemical analysis of 5 dpf uninjected and CA8 For immunoblotting, 3 dpf wild-type and CA8 antisense- knockdown larvae MO-injected larvae were homogenized with a hand-held For histochemical analysis, 5 dpf zebrafish larvae were washed pestle in a microcentrifuge tube in 50 ml of lysis buffer with PBS, fixed with 4% PFA in PBS for 3 h at room tempera- (50 mM Tris–HCl pH 7.4, 150 mM NaCl, 5 mM EDTA, 1% ture, transferred to 70% ethanol and stored at 48Cbeforebeing NP40) on ice. The lysate was centrifuged at 10 000g for embedded in paraffin. The paraffin-embedded samples were cut 5 min, 10 ml of the lysate was used per lane on a 10% SDS into 5 mmslices. polyacrylamide gel and the separated proteins were transferred The fixed sections were deparaffinized in xylene, rehydrated onto a polyvinylidene difluoride membrane (Immobilon, in an alcohol series and histologically stained with Mayer’s Millipore). The CARP VIII protein was detected by incubating hematoxylin and Eosin Y (both from Sigma-Aldrich). After the membrane with primary rabbit anti-human CARP VIII dehydration, the slides were mounted with Entellan NeuTM antibodies (Santa Cruz Biotechnology) raised against amino (Merck, Darmstadt, Germany), examined and photographed acids 1 to 100 (human CARP VIII) and horseradish using a Nikon Microphot microscope (Nikon Microphot-FXA, + peroxidase-conjugated goat anti-rabbit IgG (H L) antibody. Japan). All of these procedures were carried out at room tem- The signals were visualized using a chemiluminescence perature. system (Western Lightning; Perkin Elmer Life Science). For mRNA analysis of the MO2-injected 5 dpf morphant, larvae were homogenized along with wild type in trizol reagent, and mRNA was isolated according to the instructions in TUNEL assay on whole mount larvae RNAeasyw Mini kit (Qiagen, Hilden, Germany). The cDNA The whole mount morphant and control larvae were sectioned, was prepared according to the procedure described previously, and TUNEL assays were performed using a QIA39 FragELTM and PCR-amplified products were electrophoresed on a 0.8% DNA Fragmentation Detection Kit, Fluorescent—TdT agarose gel, visualized and photographed. Enzyme (Merck Chemicals Ltd., Nottingham, UK). The zebrafish larvae were fixed with 4% PFA in PBS for 3 h and transferred to 70% ethanol before being embedded in paraffin and Live image analysis of zebrafish phenotypes sectioned. Proteinase K-treated whole larvae or deparaffinized Gross morphology was analyzed by light-field microscopy. sections (5 mm) of larvae were incubated with the TdT Typically, 10–20 CA8-MOs injected larvae were screened enzyme followed by incubation with anti-digoxigenin. The per MO experiment, with a similar number of matched con- fluorescein, generated as intense signal, was detected by trols tested. Larvae were first anesthetized using 5% Tricaine fluorescence microscopy. 432 Human Molecular Genetics, 2013, Vol. 22, No. 3

Electron microscopic analysis 3. Jiao, Y., Yan, J., Zhao, Y., Donahue, L.R., Beamer, W.G., Li, X., Roe, B.A., Ledoux, M.S. and Gu, W. (2005) Carbonic anhydrase-related Five-day-old zebrafish larvae were fixed overnight with 2% protein VIII deficiency is associated with a distinctive lifelong gait glutaraldehyde, 2% formaldehyde in 0.1 M sodium cacodylate disorder in waddles mice. Genetics, 171, 1239–1246. buffer, pH 7.4 at +48C and post fixed with 1% reduced, buf- 4. Sly, W.S. and Hu, P.Y. (1995) Human carbonic anhydrases and carbonic anhydrase deficiencies. Annu. Rev. Biochem., 64, 375–401. fered osmium tetroxide for 1 h at room temperature. After de- 5. Pastorekova, S., Parkkila, S., Pastorek, J. and Supuran, C.T. (2004) hydration with a graded ethanol series, the specimens were Carbonic anhydrases: current state of the art, therapeutic applications and epon-embedded into TAAB embedding resin (TAAB Ltd., future prospects. J. Enzyme Inhib. Med. Chem., 19, 199–229. UK). Semithin sections were cut and stained with Toluidine 6. Bellingham, J., Gregory-Evans, K. and Gregory-Evans, C.Y. (1998) Blue for light microscopy analysis (Axiophot, Zeiss Lumar Sequence and tissue expression of a novel human carbonic anhydrase-related protein, CARP-2, mapping to chromosome 19q13.3.

V.12, Thornwood, NY, USA equipped with an Olympus Biochem. Biophys. Res. Commun., 253, 364–367. Downloaded from DP70 digital camera). A suitable area was then selected for 7. Hewett-Emmett, D. and Tashian, R.E. (1996) Functional diversity, ultrathin sectioning, and sections were collected on pioloform- conservation, and convergence in the evolution of the a-, b-, and coated single-slot copper grids and post stained with uranyl g-carbonic anhydrase gene families. Mol. Phylogenet. Evol., 5, 50–77. 8. Taniuchi, K., Nishimori, I., Takeuchi, T., Fujikawa-Adachi, K., Ohtsuki, acetate and lead citrate. The resulting sections were analyzed Y. and Onishi, S. (2002) Developmental expression of carbonic using a transmission electron microscope (Tecnai 12, FEI, anhydrase-related proteins VIII, X, and XI in the human brain. Hilsboro, OR, USA) operated at 80 kV, and images were Neuroscience, 112, 93–99. http://hmg.oxfordjournals.org/ acquired with a MultiScan 794 1K × 1K CCD camera 9. Aspatwar, A., Tolvanen, M.E. and Parkkila, S. (2010) Phylogeny and (Gatan, Pleasanton, CA, USA). In all comparisons, at least expression of carbonic anhydrase-related proteins. BMC Mol. Biol., 11,25. 10. Aspatwar, A., Tolvanen, M.E., Ortutay, C. and Parkkila, S. (2010) six larvae for each category were examined. Carbonic anhydrase related protein VIII and its role in neurodegeneration and cancer. Curr. Pharm. Des., 16, 3264–3276. 11. Zakon, H.H. (2002) Convergent evolution on the molecular level. Brain SUPPLEMENTARY MATERIAL Behav. Evol., 59, 250–261. 12. Carradice, D. and Lieschke, G.J. (2008) Zebrafish in hematology: sushi or Supplementary material is available at HMG online. science? Blood, 111, 3331–3342. 13. Lohi, O., Parikka, M. and Ramet, M. The zebrafish as a model for at Tampere University Library. Department of Health Sciences on May 19, 2014 paediatric diseases. Acta Paediatr. [Epub ahead of print]. ACKNOWLEDGEMENTS 14. Hirota, J., Ando, H., Hamada, K. and Mikoshiba, K. (2003) Carbonic anhydrase-related protein is a novel binding protein for inositol We thank Marianne Kuuslahti and Aulikki Lehmus for their 1,4,5-trisphosphate receptor type 1. Biochem. J., 372, 435–441. skillful technical assistance with the experiments. We greatly 15. Mikoshiba, K. (2007) IP3 receptor/Ca2+ channel: from discovery to new signaling concepts. J. Neurochem., 102, 1426–1446. appreciate the help by Leena Ma¨kinen and Matilda Martikai- 16. Lakkis, M.M., Bergenhem, N.C., O’Shea, K.S. and Tashian, R.E. (1997) nen in morpholino injections. We are grateful to Annemari Expression of the acatalytic carbonic anhydrase VIII gene, Car8, during Uusima¨ki for providing cDNA samples from different adult mouse embryonic development. Histochem. J., 29, 135–141. zebrafish tissues. 17. Ekker, S.C. and Larson, J.D. (2001) Morphant technology in model developmental systems. Genesis, 30, 89–93. 18. Diekmann, H., Anichtchik, O., Fleming, A., Futter, M., Goldsmith, P., Roach, A. and Rubinsztein, D.C. (2009) Decreased BDNF levels are a Conflict of Interest statement. None declared. major contributor to the embryonic phenotype of huntingtin knockdown zebrafish. J. Neurosci., 29, 1343–1349. 19. Yan, J., Jiao, Y., Jiao, F., Stuart, J., Donahue, L.R., Beamer, W.G., Li, X., FUNDING Roe, B.A., LeDoux, M.S. and Gu, W. (2007) Effects of carbonic anhydrase VIII deficiency on cerebellar gene expression profiles in the This work was supported by the competitive Research wdl mouse. Neurosci. Lett., 413, 196–201. Funding of the Tampere University Hospital (Grant 9L071, 20. Hirasawa, M., Xu, X., Trask, R.B., Maddatu, T.P., Johnson, B.A., Naggert, J.K., Nishina, P.M. and Ikeda, A. (2007) Carbonic anhydrase Grant 9M093, Grant 9L070) and grants from the Academy related protein 8 mutation results in aberrant synaptic morphology and of Finland (S.P., M.R., M.P.), Sigrid Juselius Foundation excitatory synaptic function in the cerebellum. Mol. Cell Neurosci., 35, (S.P., M.R.) and Tampere Tuberculosis Foundation (M.R. 161–170. and M.P.). The zebrafish work was carried out at the 21. Fares, M.A. and McNally, D. (2006) CAPS: coevolution analysis using protein sequences. Bioinformatics, 22, 2821–2822. Tampere Zebrafish Core Facility funded by the Biocenter 22. Sterling, D., Reithmeier, R.A. and Casey, J.R. (2001) A transport Finland, Tampere Tuberculosis Foundation and Emil Aaltonen metabolon. Functional interaction of carbonic anhydrase II and chloride/ Foundation. bicarbonate exchangers. J. Biol. Chem., 276, 47886–47894. 23. Westerfield, M. (2000) The Zebrafish Book: A Guide for the Laboratory Use of Zebrafish (Danio Rerio). University of Oregon, Eugene. REFERENCES 24. Kimmel, C.B., Ballard, B.W., Kimmel, S.R., Ullmann, B. and Schilling, T.F. (1995) Stages of embryonic development of the zebrafish. Dev. Dyn., 1. Kaya, N., Aldhalaan, H., Al-Younes, B., Colak, D., Shuaib, T., 203, 253–310. Al-Mohaileb, F., Al-Sugair, A., Nester, M., Al-Yamani, S., Al-Bakheet, 25. Pfaffl, M.W. (2001) A new mathematical model for relative quantification A. et al. (2011) Phenotypical spectrum of cerebellar ataxia associated with in real-time RT-PCR. Nucleic Acids Res., 29, e45. a novel mutation in the CA8 gene, encoding carbonic anhydrase (CA) 26. Picaud, S.S., Muniz, J.R., Kramm, A., Pilka, E.S., Kochan, G., VIII. Am. J. Med. Genet. B. Neuropsychiatr. Genet., 156, 826–834. Oppermann, U. and Yue, W.W. (2009) Crystal structure of human 2. Turkmen, S., Guo, G., Garshasbi, M., Hoffmann, K., Alshalah, A.J., carbonic anhydrase-related protein VIII reveals the basis for catalytic Mischung, C., Kuss, A., Humphrey, N., Mundlos, S. and Robinson, P.N. silencing. Proteins, 76, 507–511. (2009) CA8 mutations cause a novel syndrome characterized by ataxia 27. Berman, H.M., Westbrook, J., Feng, Z., Gilliland, G., Bhat, T.N., Weissig, and mild mental retardation with predisposition to quadrupedal gait. PLoS H., Shindyalov, I.N. and Bourne, P.E. (2000) The Protein Data Bank. Genet., 5, e1000487. Nucleic Acids Res., 28, 235–242.